Antiviral prodrugs and nanoformulations thereof

ABSTRACT

The present invention provides prodrugs and methods of use thereof.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of International Application No.PCT/US2019/057406, filed Oct. 22, 2019, which claims priority under 35U.S.C. § 119(e) to U.S. Provisional Patent Application No. 62/748,798,filed Oct. 22, 2018. The foregoing applications are incorporated byreference herein in their entireties.

STATEMENT AS TO FEDERALLY SPONSORED RESEARCH

This invention was made with government support under Grants Nos. R01MH104147, P01 DA028555, R01 NS036126, P01 NS031492, R01 NS034239, P01MH064570, P30 MH062261, P30 AI078498, R01 AG043540, and R56 AI138613awarded by the National Institutes of Health. The government has certainrights in the invention.

FIELD OF THE INVENTION

The present invention relates generally to the delivery of therapeutics.More specifically, the present invention relates to compositions andmethods for the delivery of therapeutic agents to a patient for thetreatment of a disease or disorder.

BACKGROUND OF THE INVENTION

Remarkable progress has been made in the development of effectivediagnostics and treatments against human immunodeficiency virus type one(HIV-1). Antiretroviral therapy (ART) has markedly reduceddisease-associated morbidities and mortality, enabling a nearly normalquality of life for infected people (Vittinghoff, et al. (1999) J.Infect Dis., 179(3):717-720; Lewden, et al. (2007) J. Acquir. ImmuneDefic. Syndr., 46(1):72-77). However, ART requires life-long treatmentin order to suppress viral replication and prevent AIDS onset. Moreover,the effectiveness of ART can be hampered by HIV-1 resistance, drugtoxicities, and poor patient adherence (Wensing, et al. (2014) Top.Antivir. Med., 22(3):642-650; Siliciano, et al. (2013) Curr. Opin.Virol., 3(5):487-494; Prosperi, et al. (2012) BMC Infect. Dis.,12:296-296; Van den Berk, et al. (2016) Abstract number: 948, InConference on Retroviruses and Opportunistic Infections, 22-25;Siefried, et al. (2017) PLoS One 12(4):e0174613). Treatment fatigue,lack of financial and social support, co-existing mental symptoms,and/or substance abuse can result in the failure to adhere to criticalART regimens (Tucker, et al. (2017) EBioMedicine, 17:163-171).

Long-acting parenteral (LAP) antiretroviral drugs have improved regimenadherence (Spreen, et al. (2013) Curr. Opin. HIV AIDS, 8(6):565-571).Reducing the treatment schedule from daily to monthly or evenless-frequent administration provides greater patient privacy andsatisfaction and improves regimen adherence (Sangaramoorthy, et al.(2017) J. Assoc. Nurses AIDS Care, 28(4):518-531; Carrasco, et al.(2017) Afr. J. AIDS Res. 16(1):11-18; Williams, et al. (2013) Nanomed.Lond. 8(11):1807-1813). However, only a few antiretroviral drugs havebeen successfully reformulated into LAPs.

Cabotegravir (CAB) is an integrase inhibitor or integrase strandtransfer inhibitor (INSTI) with low aqueous solubility, high meltingpoint, high potency, long half-life, and slow metabolic clearance(Karmon, et al. (2015) J. Acquir. Immune Defic. Syndr., 68(3):39-41;Trezza, et al. (2015) Curr. Opin. HIV AIDS 10(4):239-245). Theseproperties enable CAB to be formulated in a 200-mg/mL suspension (CABLAP) and administered intramuscularly monthly or even less frequently(Margolis, et al. (2017) Lancet 390(10101):1499-1510; Spreen, W. W.(2014) J. Acquir. Immune Defic. Syndr., 67(5):481-486). Notably, CABplus rilpivirine (RPV) is the first long-acting combination ART regimenwhere monthly or every other month CAB and RPV LAP formulations havedemonstrated comparable antiretroviral activity to daily oral three-drugcombinations for maintenance therapy (Margolis, et al. (2017) Lancet390(10101):1499-1510).

Advantageously, CAB is primarily metabolized by uridine diphosphateglucuronosyltransferase (UGT) 1A1 and has a low potential to interactwith other antiretroviral drugs (Trezza, et al. (2015) Curr. Opin. HIVAIDS 10(4):239-245; Bowers, et al. (2016) Xenobiotica 46(2):147-162).CAB LAP is highly protective against rectal, vaginal, and intravenoussimian/human immunodeficiency virus (SHIV) transmission in non-humanprimates and has been advanced into clinical trials for HIV prevention(NCT02720094) (Andrews, et al. (2014) Science 343(6175):1151-1154;Andrews, et al. (2015) Sci. Transl. Med., 7(270) 270ra4; Radzio, et al.(2015) Sci. Transl. Med., 7(270) 270ra5-270ra5; Andrews, et al. (2016)AIDS 2016:461-467). However, the dosing pattern of CAB LAP haslimitations. Specifically, split injections given in 2 mL volumes arerequired which leads to treatment cessations because of intolerableinjection site reactions (Margolis, et al. (2017) Lancet390(10101):1499-510; Markowitz, et al. (2017) Lancet HIV 4(8):331-340).Moreover, the maximal dosing interval is only 8 weeks. Recently, theadministration of CAB LAP every 12 weeks has been tested with the aim ofmaintaining plasma CAB concentrations above 4 timesprotein-binding-adjusted 90% inhibitory concentration (4×PA-IC₉₀, 660ng/mL), a concentration demonstrated to be protective against newinfections in macaques (Spreen, W. W. (2014) J. Acquir. Immune Defic.Syndr., 67(5):481-486; Andrews, et al. (2014) Science343(6175):1151-1154; Andrews, et al. (2015) Sci. Transl. Med., 7(270)270ra4; Radzio, et al. (2015) Sci. Transl. Med., 7(270) 270ra5-270ra5;Andrews, et al. (2016) AIDS 2016:461-467; Markowitz, et al. (2017)Lancet HIV 4(8):331-340; Spreen, et al. (2014) J. Acquir. Immune Defic.Syndr., 67(5):487-492). However, two-thirds of participants had fasterthan anticipated drug absorption leading to plasma drug concentrationsbelow the targeted effective concentration of 4×PA-IC₉₀ at 12 weeks.Thus, ways to extend the dose interval beyond 8 weeks and reduceinjection volumes to improve regimen adherence are greatly needed (Boyd,et al. (2017) Lancet 390(10101):1468-1470).

SUMMARY OF THE INVENTION

In accordance with the instant invention, prodrugs of integraseinhibitors are provided. In a particular embodiment, the prodrugcomprises an integrase inhibitor modified with an ester comprising analiphatic or alkyl group (e.g., an aliphatic or alkyl comprising about 3to about 30 carbons). In a particular embodiment, the aliphatic or alkylgroup is the alkyl chain of a fatty acid or a saturated linear aliphaticchain, optionally substituted with at least one heteroatom. In aparticular embodiment, the integrase inhibitor is selected from thegroup consisting of cabotegravir (CAB), raltegravir (RAL), elvitegravir(EVG), dolutegravir (DTG), and bictegravir (BIC). Compositionscomprising at least one prodrug of the instant invention and at leastone pharmaceutically acceptable carrier are also encompassed by thepresent invention.

In accordance with another aspect of the instant invention,nanoparticles comprising at least one prodrug of the instant inventionand at least one polymer or surfactant are provided. In a particularembodiment, the prodrug is crystalline. In a particular embodiment, thepolymer or surfactant is an amphiphilic block copolymer such as anamphiphilic block copolymer comprising at least one block ofpoly(oxyethylene) and at least one block of poly(oxypropylene) (e.g.,poloxamer 407). The nanoparticle may comprise a polymer or surfactantlinked to at least one targeting ligand. An individual nanoparticle maycomprise targeted and non-targeted surfactants. In a particularembodiment, the nanoparticles have a diameter of about 100 nm to 1Compositions comprising at least one nanoparticle of the instantinvention and at least one pharmaceutically acceptable carrier are alsoencompassed by the present invention.

In accordance with another aspect of the instant invention, methods fortreating, inhibiting, and/or preventing a disease or disorder in asubject in need thereof are provided. The methods comprise administeringto the subject at least one prodrug or nanoparticle of the instantinvention, optionally within a composition comprising a pharmaceuticallyacceptable carrier. In a particular embodiment, the disease or disorderis a viral infection (e.g., a retroviral infection). In a particularembodiment, the method further comprises administering at least onefurther therapeutic agent or therapy for the disease or disorder, e.g.,at least one additional anti-HIV compound.

BRIEF DESCRIPTIONS OF THE DRAWING

FIG. 1A provides images of NM2CAB (top) and NCAB (bottom) nanoparticlesas determined by scanning electron microscopy. FIG. 1B provides graphsof NM2CAB nanoparticle stability at 25° C. over the indicated periods oftime. Particularly, nanoparticle zeta potential (top), polydispersityindex (middle), and particle diameter (bottom) were determined bydynamic light scattering

FIG. 2A provides a graph (top) of the drug uptake as measured byUPLC-UV/Vis by human monocyte derived macrophages (MDM) and a graph(bottom) of drug retention by MDM and collected at the indicatedtimepoints for intracellular drug analysis. FIG. 2B provides a graph(top) of HIV-1 reverse transcriptase activity at the indicatedconcentration of drug and a graph (bottom) of HIV-1 reversetranscriptase activity in MDM treated with drug and challenged withHIV-1ADA and measured at the indicated timepoints post-treatment. FIG.2C provides images of p24 stained MDM cells post-challenge.

FIG. 3A provides a graph of plasma CAB levels after a singleintramuscular (IM) dose of NCAB, NMCAB or NM2CAB in female NSG (NOD scidgamma mouse) mice. Administered dose was 45 mg CAB equivalents (eq)/kg.Top bold dashed line indicates plasma CAB 4×PA-IC₉₀ of 664 ng/ml and thebottom stippled line shows the plasma CAB 1×PA-IC₉₀ of 166 ng/ml. FIG.3B provides a graph of plasma CAB levels after a single intramuscular(IM) dose of NM3CAB in female NSG (NOD scid gamma mouse) mice.Administered dose was 45 mg CAB equivalents (eq)/kg. Top bold dashedline indicates plasma CAB 4×PA-IC₉₀ of 664 ng/ml and the bottom stippledline shows the plasma CAB 1×PA-IC₉₀ of 166 ng/ml.

FIG. 4 provides images of the antiretroviral dose-response of NM2CAB at10, 50 or 100 μM concentration by immunocytochemistry for HIV-1 p24antigen.

FIG. 5 provides graphs of prodrug levels. The tissue and bloodbiodistribution of prodrugs (MCAB or M2CAB) were assessed at 14, 28, 42and 364 days after a single IM injection of NMCAB or NM2CAB. Prodruglevels were measured in the spleen, liver, gut, lung, brain, rectaltissue, kidneys, lymph nodes-anatomical associated tissues and blood.Prodrug levels were determined by LC-MS/MS. Data are expressed asmean±SEM. For day 14, 28 and 42 groups, animal numbers in each groupwere N=5, and for day 364 group, animal numbers were N=3 (NMCAB), N=4(NM2CAB). A one-way ANOVA followed by a Tukey post's test was used tocompare drug levels in tissues among three treatments (*P<0.05,**P<0.01, ***P<0.001).

FIGS. 6A and 6B provide graphs of the pharmacokinetics andbiodistribution of NM2CAB in rhesus macaques. Four rhesus macaques wereadministered with 45 mg/kg CAB-equivalent dose of NM2CAB by a single IMinjection. Plasma samples were collected and assayed for CAB (top lines)and M2CAB (bottom lines) levels up to day 393 (FIG. 6A). Rectal, lymphnode, and adipose tissue biopsies were collected at day 204 followingdrug administration and assayed for both CAB (top) and M2CAB (bottom)concentrations (FIG. 6B). Both plasma and tissue drug concentrationswere determined by LCMS/MS.

DETAILED DESCRIPTION OF THE INVENTION

Maximal restriction of viral load in tissue infectious sites canfacilitate viral eradication strategies. This can be achieved bygeneration of potent lipophilic and hydrophobic antiretroviral prodrugnanocrystals stabilized by surfactants. Hydrophobicities, drughydrolysis rates, and antiretroviral potencies must be balanced foroptimal therapeutic effect. Herein, it is demonstrated that themanipulation of the size of the hydrophobic and lipophilic carbon chainlength of a prodrug can optimize therapeutic efficacy, particularly withregard to long acting slow effective release antiretroviral therapy(LASER ART). LASER ART refers to a long acting antiretroviral druggenerated from a nanocrystal prodrug with a lipid tail. MyristoylatedCAB prodrug nanocrystal provide sustained plasma CAB concentrations atthe PA-IC₉₀ for 4 months in rhesus macaques after single 45 mg/kg CABequivalent intramuscular injection dose. Herein, it is shown thatfurther chemical modifications unexpectedly serve to enhance CABlipophilicity and hydrophobicity, improve drug potency, and slow prodrughydrolysis, thereby extensively extending the half-life of the parentdrug. The novel cabotegravir prodrugs (M2CAB) enhance drug encapsulationwith appropriate excipients and stabilizers, such as poloxamer 407(P407). M2CAB nanoformulations (NM2CAB) provide sustained drug releaseand site specific antiretroviral drug delivery. The prodrugs comprisenative drug conjugated to hydrophobic moieties via hydrolyzable covalentbonds. The NM2CAB nanoformulations were readily taken up by humanmonocyte-derived macrophages (MDM) with sustained drug retention for 30days in vitro; whereas parent drug nanoformulation (NCAB) or firstgeneration myristoylated cabotegravir (NMCAB) showed HIV-1 breakthroughin MDM within one or 20 days of treatment, respectively. Notably, MDMtreated with NM2CAB exhibited sustained antiretroviral activitiesfollowing HIV-1 challenge for up to 30 days after single drug treatment.HIV-1p24 was not detected in the NM2CAB treated group at 5 dayincremental time points for up to or greater than 30 days. Further, asingle intramuscular (IM) injection of NM2CAB at 45 mg CABequivalents/kg into female NSG (NOD scid gamma mouse) mice demonstrateda zero order controlled release kinetics of active CAB and provided druglevels at or above 4 times the PA-IC₉₀ for greater than 5 months. TheNM2CAB nanoformulations presented herein improves upon currentcombination ART regimens that require multiple daily administrations byreducing pill burden, lowering the risk of viral rebound, limitingtoxicities, and/or allowing for drug penetration into viral reservoirs.Importantly, NM2CAB also facilitates a dosing interval of once every sixmonths (or even less frequently) to maximize the effectiveness ofpre-exposure prophylaxis or treatment regimens.

Long acting slow effective release ART (LASER ART) formulations canextend dosing intervals, reduce systemic toxicity, and improvepharmacokinetic (PK) and pharmacodynamic (PD) profiles (Sillman, et al.,Nat. Commun. (2018) 9:443; Zhou, et al., Biomaterials (2018) 151:53-65;McMillan, et al., Antimicrob. Agents Chemother. (2018) 62:e01316-17).Herein, novel integrase inhibitor prodrugs, long-acting slow effectiverelease formulations thereof, and methods of synthesis and use thereofare provided. Integrase inhibitors (integrase strand transfer inhibitors(INSTIs)) are a class of antiretroviral drug designed to block theaction of integrase (e.g., HIV integrase), a viral enzyme that insertsthe viral genome into the DNA of the host cell. Examples of integraseinhibitors include, without limitation, cabotegravir (CAB, GSK1265744),raltegravir (RAL), elvitegravir (EVG), dolutegravir (DTG, GSK1349572),bictegravir (BIC, GS-9883), BI 224436 (Boehringer Ingelheim, Ingelheim,Germany), and MK-2048 (Merck, Kenilworth, N.J.). The hydrophobic andlipophilic prodrugs and their slow effective release formulationsexhibit enhanced potency and efficacy, increased cellular and tissuepenetration and extended half-lives compared to parent integraseinhibitor. The prodrugs and their formulations of the instant inventionand their combinations can be used in the management of viral (e.g.,retroviral) infections.

Treatments of viral infections, particularly HIV infections, which arecurrently available, include inhibitors of viral entry, nucleosidereverse transcriptase, nucleotide reverse transcriptase, integrase, andprotease. Resistance is linked to a shortened drug half-life, the virallife cycle, and rapid mutations resulting in a high genetic variability.Combination therapies, e.g., antiretroviral therapies (ART), which areconsidered “cocktail” therapy, have gained substantial attention.Benefits include decreased viral resistance, limited toxicities,improved adherence to therapeutic regimens and sustained antiretroviralefficacy. Combination therapies minimize potential drug resistance bysuppressing viral (e.g., HIV) replication, thereby reducing spontaneousresistant mutants. Treatment failure is attributed, in part, to theshort drug half-lives. Furthermore, failure can also be attributed, inpart, to limited drug access to tissue and cellular viral reservoirs,thereby precluding viral eradication efforts. To these ends, thedevelopment of cell and tissue targeted nanoformulated prodrug(nanoparticle) platforms are of considerable interest in the managementof viral (e.g., HIV) infections. Pre-exposure prophylaxis (PrEP) isanother strategy used in the management of viral (e.g., HIV)transmission. For example, TRUVADA® (tenofovir/emtricitabine) has beenapproved for pre-exposure prophylaxis against HIV infection.Additionally, the combination of lamivudine and zidovudine (COMBIVIR®)has been used as pre-exposure prophylaxis and post-exposure prophylaxis.

The prodrugs and nanoformulated prodrugs (nanoparticles) provided hereinunexpectedly extend the drug half-life, increase hydrophobicity andlipophilicity, and improve antiretroviral efficacy. This will benefitpeople who have to receive daily high doses or even several doses a day,since lower dosage with less dosing frequency would not only decreasethe side effects, but also be convenient to the patients. The prodrugsand nanoformulated prodrugs (nanoparticles) provided herein may also beused as a post-exposure treatment and/or pre-exposure prophylaxis (e.g.,for people who are at high risk of contracting HIV-1). In other words,the prodrugs and nanoparticles of the instant invention and theircombination may be used to prevent a viral infection (e.g., HIVinfection) and/or treat or inhibit an acute or long term viral infection(e.g., HIV infection). While the prodrugs and nanoparticles of theinstant invention are generally described as anti-HIV agents, theprodrugs and nanoformulations of the instant invention are alsoeffective against other viral infections including, without limitation:retroviruses (e.g., lentiviruses), hepatitis B virus (HBV), hepatitis Cvirus (HCV), and human T-cell leukemia viruses (HTLV), particularlyretroviruses.

The present invention describes novel, potent, broad spectrum prodrugswith improved biological activity over parent drugs. Methods for theencapsulation of the prodrugs into long acting slow effectiveformulations for efficient intracellular and tissue delivery andextended drug half-lives are also provided. The long acting sloweffective release (LASER) compositions described herein exhibit enhancedpotency and may be used as effective therapeutic or preventativeinterventions against viral infections (e.g., retroviral infections).

Prodrugs of the instant invention allow for the efficient intracellulardelivery of integrase inhibitors. Herein, prodrugs are provided whichare derivatives of integrase inhibitors wherein a chemical moiety,particularly an oxygen containing moiety such as a hydroxyl group, hasbeen replaced with an ester moiety comprising a hydrophobic andlipophilic cleavable moiety (e.g., therapeutic fatty alcohols). Thehydrophobic and lipophilic cleavable moiety (e.g., therapeutic fattyalcohols) can exhibit antiviral activity against enveloped viruses(Katz, et al., Ann. NY Acad. Sci. (1994) 724:472-88). Notably,synergistic interactions between therapeutic fatty alcohols andnucleoside analogs can substantially enhance antiviral potency of thenucleosides (Marcelletti, et al., Antiviral Res. (2002) 56:153-66).

As described herein, the prodrugs may comprise labile therapeutic fattyalcohols to improve drug potency, accelerate intracellular and tissuepenetrance, protein binding, and bioavailability. The hydrophobic natureof the synthesized prodrugs facilitates encapsulation into long actingslow release drug nanocrystals with improved biopharmaceutical features.The nanoformulations of the instant invention may be composed of prodrugparticles dispersed in sterile aqueous suspensions and stabilized bypolymeric excipients, lipids, and/or surfactants or polymers. Withoutbeing bound by theory, the mechanism of drug release involvesdissolution of the prodrug from the nanoparticle followed by efficientcleavage to generate two bioactive agents, i.e., the integrase inhibitorand the broad-spectrum antiviral fatty alcohols.

The benefits of the system described herein include, without limitation,improved drug potency, bioavailability and extended half-life forpatient convenience. Indeed, the nanoformulations described in thisinvention displayed significant increase in drug uptake bymonocyte-derived macrophages (MDM). Also, the modified drug andnanoparticles exhibited enhanced potency through increased and extendedinhibition of viral replication. Therefore, the nanoformulations of theinstant invention allow for enhancement of antiviral potency andaccelerated drug delivery to anatomical reservoirs of infection.

In accordance with the instant invention, prodrugs of integraseinhibitors are provided. In a particular embodiment, the prodrugcomprises an integrase inhibitor wherein a chemical moiety such as ahydroxyl group is replaced with an ester comprising an optionallysubstituted aliphatic or alkyl group. In a particular embodiment, theester comprises a hydrocarbon chain, particularly a hydrocarbon chain of16-20 carbon atoms in length or 18 carbon atoms in length (numberinghere is inclusive of the carbon in the C═O of the ester).

In a particular embodiment, the integrase inhibitor is selected from thegroup consisting of cabotegravir (CAB), raltegravir (RAL), elvitegravir(EVG), dolutegravir (DTG), bictegravir (BIC), BI 224436, and MK-2048. Ina particular embodiment, the integrase inhibitor is selected from thegroup consisting of cabotegravir (CAB), raltegravir (RAL), elvitegravir(EVG), dolutegravir (DTG), and bictegravir (BIC). Examples of thechemical structures of these integrase inhibitors are:

In a particular embodiment, the prodrug of the instant invention isselected from the following group or a pharmaceutically acceptable saltthereof:

wherein R is an optionally substituted aliphatic or alkyl. The aliphaticor alkyl group may be unsaturated or saturated, and may be substitutedwith at least one heteroatom (e.g., O, N, or S). In a particularembodiment, R may contain an aromatic moiety that may be substitutedwith at least one heteroatom (e.g., O, N, or S).

In a particular embodiment, the alkyl or aliphatic group is hydrophobic.In a particular embodiment, R is an optionally substituted hydrocarbonchain, particularly saturated. In a particular embodiment, R is asaturated linear aliphatic chain. In a particular embodiment, the alkylor aliphatic group comprises about 3 to about 30 carbons (e.g., in themain chain of the alkyl or aliphatic group), which may be substitutedwith at least one heteroatom (e.g., O, N, or S). In a particularembodiment, R is a C14-C21 unsaturated or saturated alkyl or aliphaticgroup, which may be substituted with at least one heteroatom (e.g., O,N, or S). In a particular embodiment, R is a C14-C19 unsaturated orsaturated alkyl or aliphatic group, which may be substituted with atleast one heteroatom (e.g., O, N, or S). In a particular embodiment, Ris a C14-C17 unsaturated or saturated alkyl or aliphatic group, whichmay be substituted with at least one heteroatom (e.g., O, N, or S). In aparticular embodiment, R is a C15-C21 unsaturated or saturated alkyl oraliphatic group, which may be substituted with at least one heteroatom(e.g., O, N, or S). In a particular embodiment, R is a C15-C19unsaturated or saturated alkyl or aliphatic group, which may besubstituted with at least one heteroatom (e.g., O, N, or S). In aparticular embodiment, R is a C15-C17 unsaturated or saturated alkyl oraliphatic group, which may be substituted with at least one heteroatom(e.g., O, N, or S). In a particular embodiment, R is a C16-C21unsaturated or saturated alkyl or aliphatic group, which may besubstituted with at least one heteroatom (e.g., O, N, or S). In aparticular embodiment, R is a C16-C19 unsaturated or saturated alkyl oraliphatic group, which may be substituted with at least one heteroatom(e.g., O, N, or S). In a particular embodiment, R is a C17-C21unsaturated or saturated alkyl or aliphatic group, which may besubstituted with at least one heteroatom (e.g., O, N, or S). In aparticular embodiment, R is a C17-C19 unsaturated or saturated alkyl oraliphatic group, which may be substituted with at least one heteroatom(e.g., O, N, or S). In a particular embodiment, R is a C17 unsaturatedor saturated alkyl or aliphatic group, which may be substituted with atleast one heteroatom (e.g., O, N, or S).

In a particular embodiment, R is the alkyl chain of a fatty acid(saturated or unsaturated), particularly a C16-C22 fatty acid, a C16-C20fatty acid, a C16-C18 fatty acid, a C18-C22 fatty acid, a C18-C20 fattyacid, or a C18 fatty acid (numbering here is inclusive of the carbon inthe C═O of the ester).

In a particular embodiment, R is a saturated linear aliphatic chain or ahydrocarbon chain of at least 14 carbons (e.g., 14 to 21 carbons inlength in the chain, 14 to 19 carbons in length in the chain, 14 to 17carbons in length in the chain, 15 to 21 carbons in length in the chain,15 to 19 carbons in length in the chain, 15 to 17 carbons in length inthe chain, or 17 carbons in length in the chain). In a particularembodiment, R is a saturated linear aliphatic chain or a hydrocarbonchain of 14, 15, 16, 17, 18, 19, 20, or 21 carbons in length,particularly 14, 15, 16, 17, 18, or 19 carbons in length, 15, 16, or 17carbons in length, or 17 carbons in length. In a particular embodiment,R is a saturated linear aliphatic chain or a hydrocarbon chain of 17carbons in length.

In a particular embodiment, the prodrug of the instant invention is:

(M2CAB) or a pharmaceutically acceptable salt thereof.

The instant invention also encompasses nanoparticles (sometimes referredto herein as nanoformulations) comprising the prodrug of the instantinvention. The nanoparticles may be used for the delivery of thecompounds to a cell or host (e.g., in vitro or in vivo). In a particularembodiment, the nanoparticle is used for the delivery of antiretroviraltherapy to a subject. The nanoparticles of the instant inventioncomprise at least one prodrug and at least one surfactant or polymer. Ina particular embodiment, the nanoparticles comprise aspectroscopic-defined surfactant/polymer:drug ratio that maintainsoptimal targeting of the drug nanoparticle to maintain a macrophagedepot. These components of the nanoparticle, along with other optionalcomponents, are described hereinbelow.

Methods of synthesizing the nanoparticles of the instant invention areknown in the art. In a particular embodiment, the methods generatenanoparticles comprising a prodrug (e.g., crystalline or amorphous)coated (either partially or completely) with a polymer and/orsurfactant. Examples of synthesis methods include, without limitation,milling (e.g., wet milling), homogenization (e.g., high pressurehomogenization), particle replication in nonwetting template (PRINT)technology, and/or sonication techniques. For example, U.S. PatentApplication Publication No. 2013/0236553, incorporated by referenceherein, provides methods suitable for synthesizing nanoparticles of theinstant invention. In a particular embodiment, the polymers orsurfactants are firstly chemically modified with targeting ligands andthen used directly or mixed with non-targeted polymers or surfactants incertain molar ratios to coat on the surface of prodrug suspensions—e.g.,by using a nanoparticle synthesis process (e.g., a crystallinenanoparticle synthesis process) such as milling (e.g., wet milling),homogenization (e.g., high pressure homogenization), particlereplication in nonwetting template (PRINT) technology, and/or sonicationtechniques, thereby preparing targeted nanoformulations. Thenanoparticles may be used with or without further purification, althoughthe avoidance of further purification is desirable for quickerproduction of the nanoparticles. In a particular embodiment, thenanoparticles are synthesized using milling and/or homogenization.Targeted nanoparticles (e.g., using ligands (optionally with highmolecular weight)) may be developed through either physically orchemically coating and/or binding on the surface of polymers orsurfactants and/or drug nanosuspensions.

In a particular embodiment, the nanoparticles of the instant inventionare synthesized by adding the prodrug (e.g., crystals) to a polymer orsurfactant solution and then generating the nanoparticles (e.g., by wetmilling or high pressure homogenization). The prodrug and polymer orsurfactant solution may be agitated prior the wet milling or highpressure homogenization.

The nanoparticles of the instant invention may be used to deliver atleast one prodrug of the instant invention to a cell or a subject(including non-human animals). The nanoparticles of the instantinvention may further comprise at least one other agent or compound,particularly a bioactive agent, particularly a therapeutic agent (e.g.,antiviral compound) or diagnostic agent, particularly at least oneantiviral or antiretroviral. In a particular embodiment, thenanoparticles of the instant invention comprise at least two therapeuticagents, particularly wherein at least one is a prodrug of the instantinvention. For example, the nanoparticle may comprise an integraseinhibitor prodrug of the instant invention and at least one othertherapeutic agent (e.g., an anti-HIV agent).

In a particular embodiment, the nanoparticles of the instant inventionare a submicron colloidal dispersion of nanosized prodrug crystalsstabilized by polymers or surfactants (e.g., surfactant-coated drugcrystals; a nanoformulation). In a particular embodiment, the prodrugmay be crystalline (solids having the characteristics of crystals),amorphous, or are solid-state nanoparticles of the prodrug that isformed as crystal that combines the drug and polymer or surfactant. In aparticular embodiment, the prodrug is crystalline. As used herein, theterm “crystalline” refers to an ordered state (i.e., non-amorphous)and/or a substance exhibiting long-range order in three dimensions. In aparticular embodiment, the majority (e.g., at least 50%, 60%, 70%, 80%,90%, 95% or more) of the prodrug and, optionally, the hydrophobicportion of the surfactant are crystalline.

In a particular embodiment, the nanoparticle of the instant invention isup to about 2 or 3 μm in diameter (e.g., z-average diameter) or itslongest dimension, particularly up to about 1 μm (e.g., about 100 nm toabout 1 μm). For example, the diameter or longest dimension of thenanoparticle may be about 50 to about 800 nm. In a particularembodiment, the diameter or longest dimension of the nanoparticle isabout 50 to about 750 nm, about 50 to about 500 nm, about 200 nm toabout 500 nm, about 200 nm to about 400 nm, or about 250 nm to about 350nm. The nanoparticles may be, for example, rod shaped, elongated rods,irregular, or round shaped. The nanoparticles of the instant inventionmay be neutral or charged. The nanoparticles may be charged positivelyor negatively.

As stated hereinabove, the nanoparticles of the instant inventioncomprise at least one polymer or surfactant. A “surfactant” refers to asurface-active agent, including substances commonly referred to aswetting agents, detergents, dispersing agents, or emulsifying agents.Surfactants are usually organic compounds that are amphiphilic.

Examples of polymers or surfactants include, without limitation,synthetic or natural phospholipids, PEGylated lipids (e.g., PEGylatedphospholipid), lipid derivatives, polysorbates, amphiphilic copolymers,amphiphilic block copolymers, poly(ethyleneglycol)-co-poly(lactide-co-glycolide) (PEG-PLGA), their derivatives,ligand-conjugated derivatives and combinations thereof. Other polymersor surfactants and their combinations that can form stablenanosuspensions and/or can chemically/physically bind to the targetingligands of HIV infectable/infected CD4+ T cells, macrophages anddendritic cells can be used in the instant invention. Further examplesof polymers or surfactants include, without limitation: 1) nonionicsurfactants (e.g., pegylated and/or polysaccharide-conjugated polyestersand other hydrophobic polymeric blocks such aspoly(lactide-co-glycolide) (PLGA), polylactic acid (PLA),polycaprolactone (PCL), other polyesters, poly(propylene oxide),poly(1,2-butylene oxide), poly(n-butylene oxide),poly(tetrahydrofurane), and poly(styrene); glyceryl esters,polyoxyethylene fatty alcohol ethers, polyoxyethylene sorbitan fattyacid esters, polyoxyethylene fatty acid esters, sorbitan esters,glycerol monostearate, polyethylene glycols, polypropyleneglycols, cetylalcohol, cetostearyl alcohol, stearyl alcohol, aryl alkyl polyetheralcohols, polyoxyethylene-polyoxypropylene copolymers, poloxamines,cellulose, methylcellulose, hydroxylmethylcellulose,hydroxypropylcellulose, hydroxypropylmethylcellulose, polysaccharides,starch and their derivatives, hydroxyethylstarch, polyvinyl alcohol(PVA), polyvinylpyrrolidone, and their combination thereof); and 2)ionic surfactants (e.g., phospholipids, amphiphilic lipids,1,2-dialkylglycero-3-alkylphophocholines, 1,2-distearoyl-sn-glecro-3-phosphocholine (DSPC),1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[carboxy(polyethyleneglycol) (DSPE-PEG), dimethylaminoethanecarbamoyl cheolesterol (DC-Chol),N-[1-(2,3-Dioleoyloxy)propyl]-N,N,N-trimethylammonium (DOTAP), alkylpyridinium halides, quaternary ammonium compounds,lauryldimethylbenzylammonium, acyl carnitine hydrochlorides,dimethyldioctadecylammonium (DDAB), n-octylamines, oleylamines,benzalkonium, cetyltrimethylammonium, chitosan, chitosan salts,poly(ethylenimine) (PEI), poly(N-isopropyl acrylamide (PNIPAM), andpoly(allylamine) (PAH), poly (dimethyldiallylammonium chloride) (PDDA),alkyl sulfonates, alkyl phosphates, alkyl phosphonates, potassiumlaurate, triethanolamine stearate, sodium lauryl sulfate, sodiumdodecylsulfate, alkyl polyoxyethylene sulfates, alginic acid, alginicacid salts, hyaluronic acid, hyaluronic acid salts, gelatins, dioctylsodium sulfosuccinate, sodium carboxymethylcellulose, cellulose sulfate,dextran sulfate and carboxymethylcellulose, chondroitin sulfate,heparin, synthetic poly(acrylic acid) (PAA), poly (methacrylic acid)(PMA), poly(vinyl sulfate) (PVS), poly(styrene sulfonate) (PSS), bileacids and their salts, cholic acid, deoxycholic acid, glycocholic acid,taurocholic acid, glycodeoxycholic acid, derivatives thereof, andcombinations thereof).

The polymer or surfactant of the instant invention may be charged orneutral. In a particular embodiment, the polymer or surfactant isneutral or negatively charged (e.g., poloxamers, polysorbates,phospholipids, and their derivatives).

In a particular embodiment, the polymer or surfactant is an amphiphilicblock copolymer or lipid derivative. In a particular embodiment, atleast one polymer or surfactant of the nanoparticle is an amphiphilicblock copolymer, particularly a copolymer comprising at least one blockof poly(oxyethylene) and at least one block of poly(oxypropylene). In aparticular embodiment, the polymer or surfactant is a triblockamphiphilic block copolymer. In a particular embodiment, the polymer orsurfactant is a triblock amphiphilic block copolymer comprising acentral hydrophobic block of polypropylene glycol flanked by twohydrophilic blocks of polyethylene glycol. In a particular embodiment,the surfactant is poloxamer 407.

In a particular embodiment, the amphiphilic block copolymer is acopolymer comprising at least one block of poly(oxyethylene) and atleast one block of poly(oxypropylene). In a particular embodiment, theamphiphilic block copolymer is a poloxamer. Examples of poloxamersinclude, without limitation, Pluronic® L31, L35, F38, L42, L43, L44,L61, L62, L63, L64, P65, F68, L72, P75, F77, L81, P84, P85, F87, F88,L92, F98, L101, P103, P104, P105, F108, L121, L122, L123, F127, 10R5,10R8, 12R3, 17R1, 17R2, 17R4, 17R8, 22R4, 25R1, 25R2, 25R4, 25R5, 25R8,31R1, 31R2, and 31R4. In a particular embodiment, the poloxamer ispoloxamer 407 (Pluronic® F127).

In a particular embodiment of the invention, the polymer or surfactantis present in the nanoparticle and/or solution to synthesize thenanoparticle (as described herein) at a concentration ranging from about0.0001% to about 10% or 15% by weight. In a particular embodiment, theconcentration of the polymer or surfactant ranges from about 0.01% toabout 15%, about 0.01% to about 10%, about 0.1% to about 10%, or about0.1% to about 6% by weight. In a particular embodiment, the nanoparticlecomprises at least about 50%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99% orhigher therapeutic agent (prodrug) by weight. In a particularembodiment, the nanoparticles comprise a defined drug:polymer/surfactantratio. In a particular embodiment, the drug:polymer/surfactant ratio(e.g., by weight) is from about 10:6 to about 1000:6, about 20:6 toabout 500:6, about 50:6 to about 200:6, or about 100:6.

As stated hereinabove, the polymer or surfactant of the instantinvention may be linked to a targeting ligand. The targeting of thenanoparticles (e.g., to macrophage) can provide for superior targeting,decreased excretion rates, decreased toxicity, and prolonged half-lifecompared to free drug or non-targeted nanoparticles. A targeting ligandis a compound that specifically binds to a specific type of tissue orcell type (e.g., in a desired target:cell ratio). For example, atargeting ligand may be used for engagement or binding of a target cell(e.g., a macrophage) surface marker or receptor which may facilitate itsuptake into the cell (e.g., within a protected subcellular organellethat is free from metabolic degradation). In a particular embodiment,the targeting ligand is a ligand for a cell surface marker/receptor. Thetargeting ligand may be an antibody or antigen-binding fragment thereofimmunologically specific for a cell surface marker (e.g., protein orcarbohydrate) preferentially or exclusively expressed on the targetedtissue or cell type. The targeting ligand may be linked directly to thepolymer or surfactant or via a linker. Generally, the linker is achemical moiety comprising a covalent bond or a chain of atoms thatcovalently attaches the ligand to the polymer or surfactant. The linkercan be linked to any synthetically feasible position of the ligand andthe polymer or surfactant. Exemplary linkers may comprise at least oneoptionally substituted; saturated or unsaturated; linear, branched orcyclic aliphatic group, an alkyl group, or an optionally substitutedaryl group. The linker may be a lower alkyl or aliphatic. The linker mayalso be a polypeptide (e.g., from about 1 to about 10 amino acids,particularly about 1 to about 5). In a particular embodiment, thetargeting moiety is linked to either or both ends of the polymer orsurfactant. The linker may be non-degradable and may be a covalent bondor any other chemical structure which cannot be substantially cleaved orcleaved at all under physiological environments or conditions.

The nanoparticles/nanoformulations of the instant invention may comprisetargeted and/or non-targeted polymers or surfactants. In a particularembodiment, the molar ratio of targeted and non-targeted polymers orsurfactants in the nanoparticles/nanoformulations of the instantinvention is from about 0.001 to 100%, about 1% to about 99%, about 5%to about 95%, about 10% to about 90%, about 25% to about 75%, about 30%to about 60%, or about 40%. In a particular embodiment, the nanoparticlecomprises only targeted polymers or surfactants. In a particularembodiment, the nanoparticles/nanoformulations of the instant inventioncomprise a folate targeted polymer or surfactant and a non-targetedversion of the polymer or surfactant. In a particular embodiment, thenanoparticles/nanoformulations of the instant invention comprisefolate-poloxamer 407 (FA-P407) and/or poloxamer 407.

Examples of targeting ligands include but are not limited to macrophagetargeting ligands, CD4+ T cell targeting ligands, dendritic celltargeting ligands, and tumor targeting ligands. In a particularembodiment, the targeting ligand is a macrophage targeting ligand. Thetargeted nanoformulations of the instant invention may comprise atargeting ligand for directing the nanoparticles to HIV tissue andcellular sanctuaries/reservoirs (e.g., central nervous system, gutassociated lymphoid tissues (GALT), CD4+ T cells, macrophages, dendriticcells, etc.). Macrophage targeting ligands include, without limitation,folate receptor ligands (e.g., folate (folic acid) and folate receptorantibodies and fragments thereof (see, e.g., Sudimack et al. (2000) Adv.Drug Del. Rev., 41:147-162)), mannose receptor ligands (e.g., mannose),formyl peptide receptor (FPR) ligands (e.g., N-formyl-Met-Leu-Phe(fMLF)), and tuftsin (the tetrapeptide Thr-Lys-Pro-Arg). Other targetingligands include, without limitation, hyaluronic acid, gp120 and peptidefragments thereof, and ligands or antibodies specific for CD4, CCR5,CXCR4, CD7, CD111, CD204, CD49a, CD29, CD19, CD20, CD22, CD171, CD33,Leis-Y, WT-1, ROR1, MUC16, MUC1, MUC4, estrogen receptor, transferrinreceptors, EGF receptors (e.g. HER2), folate receptor, VEGF receptor,FGF receptor, androgen receptor, NGR, Integrins, and GD2. In aparticular embodiment, the targeting ligand is folic acid.

As stated hereinabove, the nanoparticles of the instant invention maycomprise a further therapeutic agent. The instant invention alsoencompasses therapeutic methods wherein the prodrug and/or nanoparticlesof the instant invention are co-administered with another therapeuticagent. In a particular embodiment, the therapeutic agent is hydrophobic,a water insoluble compound, or a poorly water soluble compound,particularly when included in the nanoparticle. For example, thetherapeutic agent may have a solubility of less than about 10 mg/ml,less than 1 mg/ml, more particularly less than about 100 μg/ml, and moreparticularly less than about 25 μg/ml in water or aqueous media in a pHrange of 0-14, preferably between pH 4 and 10, particularly at 20° C.

In a particular embodiment, the therapeutic agent is an antiviral or anantiretroviral. The antiretroviral may be effective against or specificto lentiviruses. Lentiviruses include, without limitation, humanimmunodeficiency virus (HIV) (e.g., HIV-1, HIV-2), bovineimmunodeficiency virus (BIV), feline immunodeficiency virus (Hy), simianimmunodeficiency virus (SIV), and equine infectious anemia virus (EIA).In a particular embodiment, the therapeutic agent is an anti-HIV agent.An anti-HIV compound or an anti-HIV agent is a compound which inhibitsHIV (e.g., inhibits HIV replication and/or infection). Examples ofanti-HIV agents include, without limitation:

(I) Nucleoside-analog reverse transcriptase inhibitors (NRTIs). NRTIsrefer to nucleosides and nucleotides and analogues thereof that inhibitthe activity of reverse transcriptase, particularly HIV-1 reversetranscriptase. NRTIs comprise a sugar and base. Examples ofnucleoside-analog reverse transcriptase inhibitors include, withoutlimitation, adefovir dipivoxil, adefovir, lamivudine, telbivudine,entecavir, tenofovir, stavudine, abacavir, didanosine, emtricitabine,zalcitabine, and zidovudine.

(II) Non-nucleoside reverse transcriptase inhibitors (NNRTIs). NNRTIsare allosteric inhibitors which bind reversibly at anonsubstrate-binding site on reverse transcriptase, particularly the HIVreverse transcriptase, thereby altering the shape of the active site orblocking polymerase activity. Examples of NNRTIs include, withoutlimitation, delavirdine (DLV, BHAP, U-90152; Rescriptor®), efavirenz(EFV, DMP-266, SUSTIVA®), nevirapine (NVP, Viramune®), PNU-142721,capravirine (S-1153, AG-1549), emivirine (+)-calanolide A (NSC-675451)and B, etravirine (ETR, TMC-125, Intelence®), rilpivirne (RPV, TMC278,Edurant™) DAPY (TMC120), doravirine (Pifeltro™), BILR-355 BS, PHI-236,and PHI-443 (TMC-278).

(III) Protease inhibitors (PI). Protease inhibitors are inhibitors of aviral protease, particularly the HIV-1 protease. Examples of proteaseinhibitors include, without limitation, darunavir, amprenavir (141W94,AGENERASE®), tipranivir (PNU-140690, APTIVUS®), indinavir (MK-639;CRIXIVAN®), saquinavir (INVIRASE®, FORTOVASE®), fosamprenavir (LEXIVA®),lopinavir (ABT-378), ritonavir (ABT-538, NORVIR®), atazanavir(REYATAZ®), nelfinavir (AG-1343, VIRACEPT®), lasinavir(BMS-234475/CGP-61755), BMS-2322623, GW-640385X (VX-385), AG-001859, andSM-309515.

(IV) Fusion or entry inhibitors. Fusion or entry inhibitors arecompounds, such as peptides, which block HIV entry into a cell (e.g., bybinding to HIV envelope protein and blocking the structural changesnecessary for the virus to fuse with the host cell). Examples of fusioninhibitors include, without limitation, CCR5 receptor antagonists (e.g.,maraviroc (Selzentry®, Celsentri)), enfuvirtide (INN, FUZEON®), T-20(DP-178, FUZEON®) and T-1249.

(V) Integrase inhibitors (in addition to the prodrug of the instantinvention). Integrase inhibitors are a class of antiretroviral drugdesigned to block the action of integrase (e.g., HIV integrase), a viralenzyme that inserts the viral genome into the DNA of the host cell.Examples of integrase inhibitors include, without limitation,raltegravir, elvitegravir, GSK1265744 (cabotegravir), GSK1349572(dolutegravir), GS-9883 (bictegravir), and MK-2048.

Anti-HIV compounds also include maturation inhibitors (e.g., bevirimat).Maturation inhibitors are typically compounds which bind HIV gag anddisrupt its processing during the maturation of the virus. Anti-HIVcompounds also include HIV vaccines such as, without limitation, ALVAC®HIV (vCP1521), AIDSVAX®B/E (gp120), and combinations thereof. Anti-HIVcompounds also include HIV antibodies (e.g., antibodies against gp120 orgp41), particularly broadly neutralizing antibodies.

More than one anti-HIV agent may be used, particularly where the agentshave different mechanisms of action (as outlined above). For example,anti-HIV agents which are not NNRTIs may be combined with the NNRTIprodrugs of the instant invention. In a particular embodiment, theanti-HIV therapy is highly active antiretroviral therapy (HAART).

The instant invention encompasses compositions (e.g., pharmaceuticalcompositions) comprising at least one prodrug and/or nanoparticle of theinstant invention and at least one pharmaceutically acceptable carrier.As stated hereinabove, the nanoparticle may comprise more than onetherapeutic agent. In a particular embodiment, the pharmaceuticalcomposition comprises a first nanoparticle comprising a first prodrugand a second nanoparticle comprising a second prodrug, wherein the firstand second prodrugs are different. In a particular embodiment, the firstprodrug is a prodrug of the instant invention and the second prodrug isa prodrug of a non-nucleoside reverse transcriptase inhibitor (NNRTI),particularly rilpivirine (RPV). The compositions (e.g., pharmaceuticalcompositions) of the instant invention may further comprise othertherapeutic agents (e.g., other anti-HIV compounds (e.g., thosedescribed herein)).

The present invention also encompasses methods for preventing,inhibiting, and/or treating a disease or disorder. The methods compriseadministering a prodrug and/or nanoparticle of the instant invention(optionally in a composition) to a subject in need thereof. In aparticular embodiment, the disease or disorder is a viral (e.g.,retroviral) infection. Examples of viral infections include, withoutlimitation: HIV, Hepatitis B, Hepatitis C, and HTLV. In a particularembodiment, the viral infection is a retroviral or lentiviral infection,particularly an HIV infection (e.g., HIV-1).

The prodrugs and/or nanoparticles of the instant invention (optionallyin a composition) can be administered to an animal, in particular amammal, more particularly a human, in order to treat/inhibit/prevent thedisease or disorder (e.g., a retroviral infection such as an HIVinfection). The pharmaceutical compositions of the instant invention mayalso comprise at least one other therapeutic agent such as an antiviralagent, particularly at least one other anti-HIV compound/agent. Theadditional anti-HIV compound may also be administered in a separatepharmaceutical composition from the prodrugs or compositions of theinstant invention. The pharmaceutical compositions may be administeredat the same time or at different times (e.g., sequentially).

The dosage ranges for the administration of the prodrugs, nanoparticles,and/or compositions of the invention are those large enough to producethe desired effect (e.g., curing, relieving, treating, and/or preventingthe disease or disorder (e.g., HIV infection), the symptoms of it (e.g.,AIDS, ARC), or the predisposition towards it). In a particularembodiment, the pharmaceutical composition of the instant invention isadministered to the subject at an amount from about 5 μg/kg to about 500mg/kg. In a particular embodiment, the pharmaceutical composition of theinstant invention is administered to the subject at an amount greaterthan about 5 μg/kg, greater than about 50 μg/kg, greater than about 0.1mg/kg, greater than about 0.5 mg/kg, greater than about 1 mg/kg, orgreater than about 5 mg/kg. In a particular embodiment, thepharmaceutical composition of the instant invention is administered tothe subject at an amount from about 0.5 mg/kg to about 100 mg/kg, about10 mg/kg to about 100 mg/kg, or about 15 mg/kg to about 50 mg/kg. Thedosage should not be so large as to cause significant adverse sideeffects, such as unwanted cross-reactions, anaphylactic reactions, andthe like. Generally, the dosage will vary with the age, condition, sexand extent of the disease in the patient and can be determined by one ofskill in the art. The dosage can be adjusted by the individual physicianin the event of any counter indications.

The prodrugs and nanoparticles described herein will generally beadministered to a patient as a pharmaceutical composition. The term“patient” as used herein refers to human or animal subjects. Theseprodrugs and nanoparticles may be employed therapeutically, under theguidance of a physician.

The pharmaceutical compositions comprising the prodrugs and/ornanoparticles of the instant invention may be conveniently formulatedfor administration with any pharmaceutically acceptable carrier(s). Forexample, the complexes may be formulated with an acceptable medium suchas water, buffered saline, ethanol, polyol (for example, glycerol,propylene glycol, liquid polyethylene glycol and the like), dimethylsulfoxide (DMSO), oils, detergents, suspending agents, or suitablemixtures thereof, particularly an aqueous solution. The concentration ofthe prodrugs and/or nanoparticles in the chosen medium may be varied andthe medium may be chosen based on the desired route of administration ofthe pharmaceutical composition. Except insofar as any conventional mediaor agent is incompatible with the nanoparticles to be administered, itsuse in the pharmaceutical composition is contemplated.

The dose and dosage regimen of prodrugs and/or nanoparticles accordingto the invention that are suitable for administration to a particularpatient may be determined by a physician considering the patient's age,sex, weight, general medical condition, and the specific condition forwhich the nanoparticles are being administered and the severity thereof.The physician may also take into account the route of administration,the pharmaceutical carrier, and the nanoparticle's biological activity.

Selection of a suitable pharmaceutical composition will also depend uponthe mode of administration chosen. For example, the nanoparticles of theinvention may be administered by direct injection or intravenously. Inthis instance, a pharmaceutical composition comprises the prodrug and/ornanoparticle dispersed in a medium that is compatible with the site ofinjection.

Prodrugs and/or nanoparticles of the instant invention may beadministered by any method. For example, the prodrugs and/ornanoparticles of the instant invention can be administered, withoutlimitation parenterally, subcutaneously, orally, topically, pulmonarily,rectally, vaginally, intravenously, intraperitoneally, intrathecally,intracerbrally, epidurally, intramuscularly, intradermally, orintracarotidly. In a particular embodiment, the prodrug and/ornanoparticle is parenterally. In a particular embodiment, the prodrugand/or nanoparticle is administered orally, intramuscularly,subcutaneously, or to the bloodstream (e.g., intravenously). In aparticular embodiment, the prodrug and/or nanoparticle is administeredintramuscularly or subcutaneously. Pharmaceutical compositions forinjection are known in the art. If injection is selected as a method foradministering the prodrug and/or nanoparticle, steps must be taken toensure that sufficient amounts of the molecules or cells reach theirtarget cells to exert a biological effect. Dosage forms for oraladministration include, without limitation, tablets (e.g., coated anduncoated, chewable), gelatin capsules (e.g., soft or hard), lozenges,troches, solutions, emulsions, suspensions, syrups, elixirs,powders/granules (e.g., reconstitutable or dispersible) gums, andeffervescent tablets. Dosage forms for parenteral administrationinclude, without limitation, solutions, emulsions, suspensions,dispersions and powders/granules for reconstitution. Dosage forms fortopical administration include, without limitation, creams, gels,ointments, salves, patches and transdermal delivery systems.

Pharmaceutical compositions containing a prodrug and/or nanoparticle ofthe present invention as the active ingredient in intimate admixturewith a pharmaceutically acceptable carrier can be prepared according toconventional pharmaceutical compounding techniques. The carrier may takea wide variety of forms depending on the form of pharmaceuticalcomposition desired for administration, e.g., intravenous, oral, directinjection, intracranial, and intravitreal.

A pharmaceutical composition of the invention may be formulated indosage unit form for ease of administration and uniformity of dosage.Dosage unit form, as used herein, refers to a physically discrete unitof the pharmaceutical composition appropriate for the patient undergoingtreatment. Each dosage should contain a quantity of active ingredientcalculated to produce the desired effect in association with theselected pharmaceutical carrier. Procedures for determining theappropriate dosage unit are well known to those skilled in the art. In aparticular embodiment, the prodrugs and/or nanoparticles of the instantinvention, due to their long-acting therapeutic effect, may beadministered once every 1 to 12 months or even less frequently. Forexample, the nanoformulations of the instant invention may beadministered once every 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 15,18, 21, 24, or more months. In a particular embodiment, the prodrugsand/or nanoparticles of the instant invention are administered less thanonce every two months. In a particular embodiment, the prodrugs and/ornanoformulations of the prodrugs are administered once every month, onceevery two months, particularly once every three months, once every fourmonths, once every five months, once every six months, once every sevenmonths, once every eight months, once every nine months, once every tenmonths, once every eleven months, once every twelve months, or lessfrequently.

Dosage units may be proportionately increased or decreased based on theweight of the patient. Appropriate concentrations for alleviation of aparticular pathological condition may be determined by dosageconcentration curve calculations, as known in the art.

In accordance with the present invention, the appropriate dosage unitfor the administration of nanoparticles may be determined by evaluatingthe toxicity of the molecules or cells in animal models. Variousconcentrations of nanoparticles in pharmaceutical composition may beadministered to mice, and the minimal and maximal dosages may bedetermined based on the beneficial results and side effects observed asa result of the treatment. Appropriate dosage unit may also bedetermined by assessing the efficacy of the nanoparticle treatment incombination with other standard drugs. The dosage units of nanoparticlemay be determined individually or in combination with each treatmentaccording to the effect detected.

The pharmaceutical composition comprising the nanoparticles may beadministered at appropriate intervals until the pathological symptomsare reduced or alleviated, after which the dosage may be reduced to amaintenance level. The appropriate interval in a particular case wouldnormally depend on the condition of the patient.

The instant invention encompasses methods of treating a disease/disordercomprising administering to a subject in need thereof a pharmaceuticalcomposition comprising a prodrug and/or nanoparticle of the instantinvention and, preferably, at least one pharmaceutically acceptablecarrier. The instant invention also encompasses methods wherein thesubject is treated via ex vivo therapy. In particular, the methodcomprises removing cells from the subject, exposing/contacting the cellsin vitro to the nanoparticles of the instant invention, and returningthe cells to the subject. In a particular embodiment, the cells comprisemacrophage. Other methods of treating the disease or disorder may becombined with the methods of the instant invention may beco-administered with the pharmaceutical compositions of the instantinvention.

The instant also encompasses delivering the nanoparticle of the instantinvention to a cell in vitro (e.g., in culture). The nanoparticle may bedelivered to the cell in at least one carrier.

Definitions

The following definitions are provided to facilitate an understanding ofthe present invention.

The singular forms “a,” “an,” and “the” include plural referents unlessthe context clearly dictates otherwise.

“Pharmaceutically acceptable” indicates approval by a regulatory agencyof the Federal or a state government or listed in the U.S. Pharmacopeiaor other generally recognized pharmacopeia for use in animals, and moreparticularly in humans.

A “carrier” refers to, for example, a diluent, adjuvant, preservative(e.g., Thimersol, benzyl alcohol), anti-oxidant (e.g., ascorbic acid,sodium metabisulfite), solubilizer (e.g., polysorbate 80), emulsifier,buffer (e.g., Tris HCl, acetate, phosphate), antimicrobial, bulkingsubstance (e.g., lactose, mannitol), excipient, auxiliary agent orvehicle with which an active agent of the present invention isadministered. Pharmaceutically acceptable carriers can be sterileliquids, such as water and oils, including those of petroleum, animal,vegetable or synthetic origin. Water or aqueous saline solutions andaqueous dextrose and glycerol solutions are preferably employed ascarriers, particularly for injectable solutions. Suitable pharmaceuticalcarriers are described in “Remington's Pharmaceutical Sciences” by E. W.Martin (Mack Publishing Co., Easton, Pa.); Gennaro, A. R., Remington:The Science and Practice of Pharmacy, (Lippincott, Williams andWilkins); Liberman, et al., Eds., Pharmaceutical Dosage Forms, MarcelDecker, New York, N.Y.; and Kibbe, et al., Eds., Handbook ofPharmaceutical Excipients, American Pharmaceutical Association,Washington.

The term “prodrug” refers to a compound that is metabolized or otherwiseconverted to a biologically active or more active compound or drug,typically after administration. A prodrug, relative to the drug, ismodified chemically in a manner that renders it, relative to the drug,less active, essentially inactive, or inactive. However, the chemicalmodification is such that the corresponding drug is generated bymetabolic or other biological processes, typically after the prodrug isadministered.

The term “treat” as used herein refers to any type of treatment thatimparts a benefit to a patient afflicted with a disease, includingimprovement in the condition of the patient (e.g., in one or moresymptoms), delay in the progression of the condition, etc. In aparticular embodiment, the treatment of a retroviral infection resultsin at least an inhibition/reduction in the number of infected cellsand/or detectable viral levels.

As used herein, the term “prevent” refers to the prophylactic treatmentof a subject who is at risk of developing a condition (e.g., HIVinfection) resulting in a decrease in the probability that the subjectwill develop the condition.

A “therapeutically effective amount” of a compound or a pharmaceuticalcomposition refers to an amount effective to prevent, inhibit, treat, orlessen the symptoms of a particular disorder or disease. The treatmentof a microbial infection (e.g., HIV infection) herein may refer tocuring, relieving, and/or preventing the microbial infection, thesymptom(s) of it, or the predisposition towards it.

As used herein, the term “therapeutic agent” refers to a chemicalcompound or biological molecule including, without limitation, nucleicacids, peptides, proteins, and antibodies that can be used to treat acondition, disease, or disorder or reduce the symptoms of the condition,disease, or disorder.

As used herein, the term “small molecule” refers to a substance orcompound that has a relatively low molecular weight (e.g., less than4,000, less than 2,000, particularly less than 1 kDa or 800 Da).Typically, small molecules are organic, but are not proteins,polypeptides, or nucleic acids, though they may be amino acids ordipeptides.

The term “antimicrobials” as used herein indicates a substance thatkills or inhibits the growth of microorganisms such as bacteria, fungi,viruses, or protozoans.

As used herein, the term “antiviral” refers to a substance that destroysa virus and/or suppresses replication (reproduction) of the virus. Forexample, an antiviral may inhibit and or prevent: production of viralparticles, maturation of viral particles, viral attachment, viral uptakeinto cells, viral assembly, viral release/budding, viral integration,etc.

As used herein, the term “highly active antiretroviral therapy” (HAART)refers to HIV therapy with various combinations of therapeutics such asnucleoside reverse transcriptase inhibitors, non-nucleoside reversetranscriptase inhibitors, HIV protease inhibitors, and fusioninhibitors.

As used herein, the term “amphiphilic” means the ability to dissolve inboth water and lipids/apolar environments. Typically, an amphiphiliccompound comprises a hydrophilic portion and a hydrophobic portion.“Hydrophobic” designates a preference for apolar environments (e.g., ahydrophobic substance or moiety is more readily dissolved in or wettedby non-polar solvents, such as hydrocarbons, than by water).“Hydrophobic” compounds are, for the most part, insoluble in water. Asused herein, the term “hydrophilic” means the ability to dissolve inwater.

As used herein, the term “polymer” denotes molecules formed from thechemical union of two or more repeating units or monomers. The term“block copolymer” most simply refers to conjugates of at least twodifferent polymer segments, wherein each polymer segment comprises twoor more adjacent units of the same kind.

An “antibody” or “antibody molecule” is any immunoglobulin, includingantibodies and fragments thereof (e.g., scFv), that binds to a specificantigen. As used herein, antibody or antibody molecule contemplatesintact immunoglobulin molecules, immunologically active portions of animmunoglobulin molecule, and fusions of immunologically active portionsof an immunoglobulin molecule.

As used herein, the term “immunologically specific” refers toproteins/polypeptides, particularly antibodies, that bind to one or moreepitopes of a protein or compound of interest, but which do notsubstantially recognize and bind other molecules in a sample containinga mixed population of antigenic biological molecules.

As used herein, the term “targeting ligand” refers to any compound whichspecifically binds to a specific type of tissue or cell type,particularly without substantially binding other types of tissues orcell types. Examples of targeting ligands include, without limitation:proteins, polypeptides, peptides, antibodies, antibody fragments,hormones, ligands, carbohydrates, steroids, nucleic acid molecules, andpolynucleotides.

The term “aliphatic” refers to a non-aromatic hydrocarbon-based moiety.Aliphatic compounds can be acyclic (e.g., linear or branched) or cyclicmoieties (e.g., cycloalkyl) and can be saturated or unsaturated (e.g.,alkyl, alkenyl, and alkynyl). Aliphatic compounds may comprise a mostlycarbon main chain (e.g., 1 to about 30 carbons) and comprise heteroatomsand/or substituents (see below). The term “alkyl,” as employed herein,includes saturated or unsaturated, straight or branched chainhydrocarbons containing 1 to about 30 carbons in the normal/main chain.The hydrocarbon chain of the alkyl groups may be interrupted with one ormore heteroatom (e.g., oxygen, nitrogen, or sulfur). An alkyl (oraliphatic) may, optionally, be substituted (e.g. with fewer than about8, fewer than about 6, or 1 to about 4 substituents). The term “loweralkyl” or “lower aliphatic” refers to an alkyl or aliphatic,respectively, which contains 1 to 3 carbons in the hydrocarbon chain.Alkyl or aliphatic substituents include, without limitation, alkyl(e.g., lower alkyl), alkenyl, halo (such as F, Cl, Br, I), haloalkyl(e.g., CCl₃ or CF₃), alkoxyl, alkylthio, hydroxy, methoxy, carboxyl,oxo, epoxy, alkyloxycarbonyl, alkylcarbonyloxy, amino, carbamoyl (e.g.,NH₂C(═O)— or NHRC(═O)—, wherein R is an alkyl), urea (—NHCONH₂),alkylurea, aryl, ether, ester, thioester, nitrile, nitro, amide,carbonyl, carboxylate and thiol. Aliphatic and alkyl groups having atleast about 5 carbons in the main chain are generally hydrophobic,absent extensive substitutions with hydrophilic substituents.

The term “aryl,” as employed herein, refers to monocyclic and bicyclicaromatic groups containing 6 to 10 carbons in the ring portion. Examplesof aryl groups include, without limitation, phenyl or naphthyl, such as1-naphthyl and 2-naphthyl, or indenyl. Aryl groups may optionallyinclude one to three additional rings fused to a cycloalkyl ring or aheterocyclic ring. Aryl groups may be optionally substituted throughavailable carbon atoms with, for example, 1, 2, or 3 groups selectedfrom hydrogen, halo, alkyl, polyhaloalkyl, alkoxy, alkenyl,trifluoromethyl, trifluoromethoxy, alkynyl, aryl, heterocyclo, aralkyl,aryloxy, aryloxyalkyl, aralkoxy, arylthio, arylazo, heterocyclooxy,hydroxy, nitro, cyano, sulfonyl anion, amino, or substituted amino. Thearyl group may be a heteroaryl. “Heteroaryl” refers to an optionallysubstituted, mono-, di-, tri-, or other multicyclic aromatic ring systemthat includes at least one, and preferably from 1 to about 4, sulfur,oxygen, or nitrogen heteroatom ring members. Heteroaryl groups can have,for example, from about 3 to about 50 carbon atoms (and all combinationsand subcombinations of ranges and specific numbers of carbon atomstherein), with from about 4 to about 10 carbons being preferred.

The following examples provide illustrative methods of practicing theinstant invention and are not intended to limit the scope of theinvention in any way.

Example 1

Maximal reduction of residual HIV-1 from its tissue sanctuary sites thatinclude brain, lymph nodes, bone marrow, gut-associated lymphoid tissueand the genital tracts can be achieved by development of long actingreservoir targeted medicines. In addition to the benefit of infrequentdosing intervals, long acting injectable drug formulations can bedesigned to utilize receptor mediated processes to achieve improved celltargeting, extended drug half-life, and enhanced tissue biodistribution.CAB is a potent viral integrase inhibitor and has been formulated as aLAP (CAB-LAP) which demonstrates sustained plasma drug levels in humansafter single intramuscular dose. Long-acting injectable nanoformulationsof rilpivirine and CAB-LAP have already enabled once-monthly injectionfor HIV suppression and prevention (Andrews, et al. (2014) Science343(6175):1151-1154; Cohen, J. (2014) Science 343(6175):1067; Spreen, etal. (2013) Curr. Opin. HIV AIDS, 8(6):565-571). The main limitations ofexisting nanoformulations include requirement for high doses and highinjection volumes. To this end, long acting slow effective releaseantiretroviral therapies (LASER ART) have been developed by synthesizinglipophilic and hydrophobic prodrug nanocrystals that permit rapid drugpenetration across physiological barriers. LASER ART maximizes drugloading with limited excipient usage, while maintaining scalability andlong-term storage. Myristoylated prodrugs have been formulated withpoloxamer surfactants. Improved potency, bioavailability, and tissuedistribution of CAB was demonstrated by increasing drug lipophilicitythat sustained plasma CAB concentrations at the PA-IC₉₀ for 4 months inrhesus macaques after single 45 mg/kg CAB equivalent intramuscularinjection. Here, improved prodrugs and nanoformulations have beensynthesized which reduce dosing frequency while improving viralreservoir targeting and drug activity.

A potent ester prodrug of CAB is provided herein which hasphysicochemical properties which allows the use of a LASER ARTformulation for infrequent administration, such as once every six totwelve months dosing intervals or even less frequently. The criteriaevaluated in selecting an optimal CAB prodrug candidate included drugpotency, lipophilicity profile, efficient in vivo conversion to CAB withminimal systemic prodrug circulation, and sustained CAB concentrationsfour times above the PA-IC₉₀ for periods of six months or longer after asingle intramuscular injection of the prodrug formulation. Surprisingly,the instant invention has demonstrated that variation in the hydrocarbonchain length of the fatty ester prodrug dramatically improves activedrug release and retention. This culminated in the identification ofM2CAB, an 18-carbon fatty ester prodrug of CAB, with unexpectedlysuperior controlled release kinetics of CAB when compared to MCAB orother fatty acid hydrocarbon chain lengths.

The prodrugs of the instant invention are derivatives of an integraseinhibitor such as CAB conjugated to hydrophobic cleavable moieties.Thus, the hydrophobic parent compound is converted into a morehydrophobic ester derivative. This is achieved through attachment of afatty acid moiety that can improve drug protein binding andbioavailability. The ester linkage between the integrase inhibitor(e.g., CAB) and derivatizing moieties is prone to enzymatic orhydrolytic cleavage. The mechanism of drug release from the particleinvolves dissolution of the prodrug from the excipient followed byefficient ester degradation to generate the active parent compound. Thedeveloped NM2CAB significantly improved drug uptake by MDM withsustained drug retention over a 30-day observation period; whereas NCABor NMCAB formulations were eliminated from macrophages after a singleday or 20 days of treatment, respectively. Similarly, MDM treated withNM2CAB exhibited enhanced and sustained antiretroviral activitiescompared to NCAB or NMCAB when challenged with HIV-1 for up to 30 daysafter single drug treatment. HIV-1p24 was not detected in the NM2CABtreated group at any of these time points. The benefits of the systeminclude unexpectedly improved drug bioavailability and extendedhalf-life. NM2CAB prolonged plasma and tissue CAB concentrationsdemonstrate that an effective once every six-months dosing interval canbe achieved.

Synthesis of M2CAB

Synthesis of M2CAB prodrugs was performed as depicted:

Briefly, the preparation of the M2CAB prodrug was performed by: 1)deprotonation of the phenol functional group with a suitable base suchas N,N-diisopropylethylamine; and 2) reaction with either acyl chlorideor activated carboxylic acid of the alkyl fatty acid.

Both steps 1 and 2 were performed in a single vessel. Specifically, thehydroxyl group was deprotonated using the appropriate reagent. Thealcohol anion was then coupled with the fatty acyl chloride or activatedcarboxylic acid to generate the prodrugs. Examples of coupling reagentsthat can be used to activate the carboxylic acid include, withoutlimitation, uranium salts, carbodiimide reagents, and phosphonium salts.An example of the base that can be used in the coupling reaction is,without limitation, N,N diisopropylethylamine (DIEA). Examples of polaraprotic solvents that can be used include, without limitation,N,N-dimethylformamide (DMF), tetrahydrofuran (THF), and acetonitrile.The reagents were mixed at 0° C. and gradually warmed to temperatureover 12-24 hours. The final compounds were purified on a silica columnchromatography and characterized by nuclear magnetic resonancespectroscopy and high performance liquid chromatography in tandem withmass spectrometry.

Hydroxyl Group Deprotonation and Coupling to Fatty Acid

A solution of CAB (2 g, 4.93 mmol, 1.0 equiv.) in anhydrousdimethylformamide (20 mL) was cooled to 0° C. under argon. N,Ndiisopropylethylamine (1.7 mL, 9.86 mmol, 2.0 equiv.) was then addeddropwise to the precooled solution of the drug. Stearoyl chloride (3.3mL, 9.86 mmol, 2.0 equiv.) was then added to the deprotonated phenolsolution. The mixture was gradually warmed to room temperature understirring over 16 hours, concentrated, and purified by flashchromatography eluting with 80 to 90% EtOAc/Hex to give the prodrug in achemical yield of 90%. The ¹H-NMR spectrum of M2CAB showing the presenceof a broad peak at 1.20-1.50 ppm and those corresponding to thealiphatic protons on the fatty acid moiety.

Formulation Synthesis

M2CAB nanocrystals were coated with poloxamer 407 (P407),1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC),1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[carboxy(polyethyleneglycol)-2000 (DSPE-PEG), and/or polyvinyl alcohol (PVA). Thenanocrystals may also be stabilized with polysorbate and polyethyleneglycol surfactants. Based on proton NMR spectroscopy data, a drug tosurfactant ratio of 100:6 by weight was used to manufacturenanoformulated M2CAB, MCAB and CAB. Briefly, 1-5% (w/v) M2CAB, MCAB orCAB and 0.06-0.3% (w/v) P407 were mixed in endotoxin free water. Thepremixed suspensions were formulated by wet milling or high-pressurehomogenization at 20,000 psi pressure until desirable size andpolydispersity index (PDI) were achieved. Nanoformulations werecharacterized for particle size, polydispersity index (PDI) and zetapotential by dynamic light scattering (FIG. 1). This was done using aMalvern Zetasizer, Nano Series Nano-ZS (Malvern Instruments Inc,Westborough, Mass.). Nanoparticle morphology was determined by scanningelectron microscopy (SEM). UPLC MS/MS was used for drug quantitation.

Macrophage Uptake and Retention

Human monocytes were obtained by leukapheresis from HIV-1/2 andhepatitis B seronegative donors and then purified by counter-currentcentrifugal elutriation (Balkundi et al., Intl. J. Nanomed. (2011)6:3393-3404; Nowacek et al., Nanomed. (2009) 4(8):903-917). Humanmonocytes were plated in a 12-well plate at a density of 1.0×10⁶ cellsper well using DMEM supplemented with 10% heat-inactivated pooled humanserum, 1% glutamine, 10 μg/mL ciprofloxacin, and 50 μg/mL gentamicin.Cells were maintained at 37° C. in a 5% CO₂ incubator. After 7-10 daysof differentiation in the presence of 1000 U/mL recombinant humanmacrophage colony stimulating factor (MCSF), MDM were treated with arange of nanoformulations and native drugs. Uptake of drug was assessedby measurements of intracellular drug concentrations at varioustimepoints after treatment. For drug retention studies, cells weretreated for 8 hours then washed with PBS and maintained with half-mediachanges every other day until collection at the indicated timepoints.For both studies, adherent MDM were washed with PBS (3×1 mL), thenscraped into 1 mL of fresh PBS, and counted at indicated time pointsusing a Countess™ automated cell counter (Invitrogen, Carlsbad, Calif.).Cells were pelleted by centrifugation at 950×g for 8 minutes at 4° C.The cell pellet was reconstituted in 200 μl of high performance liquidchromatography (HPLC)-grade methanol and probe sonicated followed bycentrifugation at 20,000×g for 20 minutes. The supernatant was analyzedfor drug content using HPLC.

As seen in FIG. 2A, NM2CAB was taken up by MDM to statistically higherlevels than NMCAB. Moreover, MDM retained NM2CAB at statistically higherlevels for longer periods of time than NMCAB (FIG. 2A).

Antiretroviral Activities

Antiretroviral efficacy was determined by measurements of HIV reversetranscriptase (RT) activity (FIGS. 2B and 2C). To assess antiretroviralefficacy, MDM were treated with either 100 μM CAB-LAP, NMCAB or NM2CABfor 8 hours. After treatment, cells were washed with PBS to removeexcess of free drug and nanoparticles and the cells were cultured withfresh media, with half-media exchanges every other day. The MDM werechallenged with HIV-1_(ADA) at a MOI of 0.01 infectious viralparticles/cell for up to 30 days. Progeny virion production was measuredby RT activity in culture medium (Kalter, et al. (1992) J. Clin.Microbiol., 30(4):993-995). HIV-1 p24 protein antigen expression wasassessed (Guo, et al. (2014) J. Virol., 88(17):9504-9513). The MDM werewashed with PBS and fixed with 4% paraformaldehyde for 15 minutes atroom temperature. The cells were blocked using 10% BSA containing 1%Triton X-100 in PBS for 30 minutes at room temperature. Followingblocking, cells were incubated with HIV-1 p24 mouse monoclonalantibodies (1:50; Dako, Carpinteria, Calif., USA) for overnight at 4°C., followed by 1 hour incubation at room temperature. HRP-labeledpolymer anti-mouse secondary antibody (Dako EnVision® System) was added(one drop/well). Hematoxylin was added to counterstain the nuclei andimages were captured using a Nikon TE300 microscope with a 20×objective. As seen in FIGS. 2B and 2C, unexpectedly superiorantiretroviral efficacy was observed for nanoformulated M2CAB comparedto nanoformulated CAB or MCAB.

Pharmacokinetic/Biodistribution Study

Female NSG mice were administered a single IM 45 mg/kg CAB equivalentdose of NM2CAB, NMCAB or NCAB. Drug levels from plasma and tissues wereassayed by UPLC-MS/MS. Drug levels in plasma were monitored weekly. Asingle IM injection of NM2CAB demonstrates zero order controlled releasekinetics of active CAB that remain four times above the PA-IC₉₀ whencompared to NMCAB or NCAB (FIG. 3). At day 364 after injection, plasmaCAB levels were at 345.2 ng/ml for NM2CAB, 8.5 ng/ml for NMCAB, andundetectable values for NCAB (limit of detection of 0.5 ng/ml).

Mice were also administered a single IM 45 mg/kg CAB equivalent dose ofNM3CAB (C22). Drug levels in plasma were monitored weekly. At day 28after injection, plasma CAB levels were at 233.2 ng/ml (FIG. 3B).Accordingly, it is clear that NM2CAB (C18) was statistically superior tothe shorter NMCAB (C14) and the longer NM3CAB (C22) for maintaining longterm, effective release of drug.

The hydrophobicity of CAB was greatly improved upon derivatization intoM2CAB prodrug. The improved hydrophobicity of M2CAB facilitatedproduction of stable formulations with high drug loading capacity.Moreover, the conversion of CAB into the more hydrophobic M2CAB andnanoparticle formation significantly improved intracellular accumulationof the drug compared to nanoformulated CAB or MCAB. Significantimprovements in MDM retention and antiretroviral efficacy were alsoobserved for nanoformulated M2CAB compared to nanoformulated CAB orMCAB. A single IM injection of NM2CAB at 45 mg CAB equivalents/kg infemale NSG mice also unexpectedly demonstrated plasma CAB concentrationsfour times above the PA-IC₉₀ for more than five months, which issignificantly greater than nanoformulated CAB or MCAB.

Example 2

Current antiretroviral drug (ARV) therapeutic regimens are both potentand well-tolerated enabling sustained, life-long suppression of humanimmunodeficiency virus type one (HIV-1) (Fauci, et al., JAMA (2019)321:844-845). However, such control of viral replication must be linkedto regimen adherence, which in turn, is affected by concurrentco-morbidity, social stigma, behavior, concurrent illicit drug-use andcost (Fauci, et al., JAMA (2019) 321:844-845). Nevertheless, even strictadherence to daily dosing commonly lead to drug toxicities, drug-druginteractions and the emergence of viral-drug resistance. All drugregimens preclude viral elimination (Dash, et al., Nat. Commun. (2019)10:2753). This highlights the fact that all therapies require life-longadherence to sustain HIV-1 suppression and mitigate disease. These haveled scientists to develop long acting (LA) therapeutic approaches. Allfocus on improving regimen adherence and drug potency (Gendelman, etal., Trends Microbiol. (2019) 27:593-606). The two most active and thosenearing Food and Drug Administration USA clinical approvals are ARV LAinjectables while implantable drug devices remain in development(Margolis, et al., Lancet (2017) 390:1499-1510; Taylor, et al., TopicsAntiviral Med. (2019) 27:50-68).

Studies performed, to date, have held considerable promise for LAinjectable for wide spread human use (Margolis, et al., Lancet (2017)390:1499-1510; Taylor, et al., Topics Antiviral Med. (2019) 27:50-68;Kerrigan, et al., PloS One (2018) 13:e0190487). As a result, LAinjectable nanoformulations of cabotegravir (CAB) and rilpivirine (RPV)as a two-drug combination will see approval for monthly administrationlikely by the end of 2019 (Margolis, et al., Lancet (2017)390:1499-1510; Taylor, et al., Topics Antiviral Med. (2019) 27:50-68;Kerrigan, et al., PloS One (2018) 13:e0190487). The “AntiretroviralTherapy as Long-Acting Suppression” (ATLAS) and “First Long-ActingInjectable Regimen” (FLAIR) both demonstrated promising safety, efficacyand tolerability (Taylor, et al., Topics Antiviral Med. (2019)27:50-68). Combination treatment affirmed non-inferiority when treatmentwas compared against standard oral three-drug regimens (Taylor, et al.,Topics Antiviral Med. (2019) 27:50-68). Likewise, drug implants havealso shown promise (Kovarova, et al., Nat. Commun. (2018) 9:4156;Gunawardana, et al., Antimicrob. Agents Chemother. (2015) 59:3913-3919;Flexner, C., Curr. Opin. HIV AIDS (2018) 13:374-380; Barrett, et al.,Antimicrob. Agents Chemother. (2018) 62(10):e01058-18.). However,limitations abound for both approaches including injection sitereactions, large injection volumes, dosage frequency, limited penetranceinto viral reservoirs (Margolis, et al., Lancet (2017) 390:1499-1510;Markowitz, et al., Lancet HIV (2017) 4:e331-e340; Zhou, et al.,Biomaterials (2018) 151:53-65). Moreover, these newer therapeuticapproaches require frequent professional health care services either byproviding the injections themselves or performing implant insertions andremovals. Measurements of tissue and plasma drug-levels correlates LAARV efficacy that include drug penetrance into mucosal, lymphoid tissuesand the central nervous system as well as long-term safety. Based on theextent of these unknowns, there is an immediate need for furtherimprovements in LA ARV regimens. Any future LA ARVs need to beadministered in reduced volumes without systemic toxicity. If achieved,ARV regimens could also behave in manners reflective of an ARV vaccinemimetic. Success would prevent new infections and reduce newtransmission, and in such manners could achieve a functional HIV-1 cure.

To such ends, LA ARV libraries have been created for a spectrum ofantiretroviral agents (Zhou, et al., Biomaterials (2018) 151:53-65;Hilaire, et al., J. Control Release (2019) 311-312:201-211; Ibrahim, etal., Int. J. Nanomedicine (2019) 14:6231-6247; Lin, et al., Chem.Commun. (Camb) (2018) 54:8371-8374; McMillan, et al., Antimicrob. AgentsChemother. (2017) 62: e01316-17; McMillan, et al., AIDS (2019)33:585-588; Sillman, et al., Nat. Commun. (2018) 9:443; Smith, et al.,Biomaterials (2019) 223:119476; Soni, et al., Biomaterials (2019)222:119441). Herein, CAB prodrugs were created with the aim ofprolonging the drug's apparent half-life and antiretroviral activitieswhile exerting tight control of hydrolysis. The synthesis andcomprehensive physiochemical characterization of three prodrugs of CABwith 14, 18 and 22 carbon linker arms (MCAB, M2CAB, and M3CAB,respectively) with complete assessment of their respectivenanoformulations (NMCAB, NM2CAB, and NM3CAB) are reported. Theseanalyses extended prior testing of the first generation CAB prodrug,MCAB (Zhou, et al., Biomaterials (2018) 151:53-65; McMillan, et al.,AIDS (2019) 33:585-588). The C18 nanoformulation, NM2CAB, enhanceduptake and retention of CAB in monocyte-macrophages and showed long-termmonthly protection against HIV-1 infectious challenge. NM2CAB generatedCAB plasma concentrations above 90% of the protein associated inhibitorconcentration (PA-IC₉₀) of 166 ng/mL for 52 weeks. This correlated withsignificant lymphoid, mucosal and gut biodistribution levels after asingle parenteral injection. There were no recorded systemic adverseevents. Parallel drug plasma concentrations in drug injected normal andimmunodeficient mice and rhesus monkeys affirmed the long-sustained drugrelease for prevention and treatment regimens. The results takentogether indicate that the prodrugs can be used for the same purpose ofa preventative vaccine against HIV-1.

Materials and Methods

Reagents

CAB was purchased from BOC sciences (Shirley, N.Y.). Pyridine,dimethylformamide (DMF), N,N-diisopropylethylamine (DIEA), myristoylchloride, stearoyl chloride, behenic acid, poloxamer 407 (P407),ciprofloxacin, dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide(MTT), dimethyl sulfoxide (DMSO), paraformaldehyde (PFA), and3,3′-diaminobenzidine (DAB) were purchased from Sigma-Aldrich (St.Louis, Mo.). Diethyl ether, ethyl acetate, hexanes, acetonitrile (ACN),methanol, LC-MS-grade water, cell culture grade water (endotoxin-free),gentamicin, potassium phosphate monobasic (KH₂PO₄), bovine serum albumin(BSA), Triton™ X-100, and TRIzol® reagent were purchased from FisherScientific (Hampton, N.H.). Monoclonal mouse anti-human HIV-1 p24 (cloneKal-1), and the polymer-based HRP-conjugated anti-mouse EnVision™+secondary were purchased from Dako (Carpinteria, Calif.).Heat-inactivated pooled human serum was purchased from InnovativeBiologics (Herndon, Va.). Dulbecco's Modification of Eagle's Medium(DMEM) was purchased from Corning Life Sciences (Tewksbury, Mass.).

Synthesis and Characterization of CAB Prodrugs

A series of three prodrugs was synthesized by esterification of the10-hydroxyl group on CAB yielding lipophilic prodrugs with 14, 18 and 22carbon linkers named MCAB, M2CAB, and M3CAB. Initially, CAB was driedfrom anhydrous pyridine and then suspended in anhydrous DMF. The mixturewas cooled to 0° C. under argon. DIEA (2 equivalents) was used todeprotonate the 10-hydroxyl group of CAB, which was further reacted with2 equivalents myristoyl chloride or stearoyl chloride for 24 hours toobtain MCAB or M2CAB respectively. M3CAB synthesis was a two-stepprocess. In the first step, behenyl chloride was synthesized bychlorination of the carboxylic acid group of behenic acid in anhydrouschloroform using 4 equivalents thionyl chloride. CAB dried fromanhydrous pyridine and resuspended in anhydrous DMF in the presence of 4equivalents trietheylamine was further added to the behenyl chloride inDMF at 0° C. under argon followed by heating the reaction at 50° C. for24 hours. All the resultant prodrugs were purified by silica gel columnchromatography using an initial mobile phase of 4:1 ethyl acetate:hexanes for initial fractions, then 9:1 ethyl acetate:hexanes for theremainder. Finally, the prodrugs were precipitated and washed in diethylether, dried under vacuum to obtain a white powder with average yield of85-95%. Successful synthesis of prodrugs was confirmed by proton andcarbon nuclear magnetic resonance (¹H and ¹³C NMR) spectra recorded byBruker Avance-III HD (Billerica, Mass.) operating at 500 MHz, a magneticfield strength of 11.7 T. Fourier Transform Infrared analysis (FT-IR)was performed on a universal attenuated total reflectance (UATR)Spectrum Two (PerkinElmer, Waltham, Mass.). Comparative crystallographicanalyses of CAB and prodrugs by powder X-ray diffraction (XRD) werecarried out in the 20 range of 2−70° at a rate of 1°/s using PANalyticalEmpyrean diffractometer (PANalytical Inc., Westborough, Mass.) withCu-Kα radiation (1.5418 Å) at 40 kV, 45 mA setting. Molecular mass wasdetermined by direct infusion into a Waters TQD mass spectrometer.

UPLC-UV/Vis quantification of CAB, MCAB, M2CAB, and M3CAB

Waters ACQUITY ultra-performance liquid chromatography (UPLC) H-Classsystem with TUV detector and Empower 3 software (Milford, Mass.) wasused to measure drug concentrations. CAB, MCAB, M2CAB, and M3CAB sampleswere separated on a Phenomenex Kinetex 5 μm C18 column (150×4.6 mm)(Torrance, Calif.). CAB was detected at 254 nm, using a mobile phaseconsisting of 65% 50 mM potassium phosphate monobasic (KH₂PO₄), pH 3.2,and 35% acetonitrile (ACN) and a flow rate of 1.0 mL/minute. MCAB,M2CAB, and M3CAB were detected at 230 nm, using a mobile phaseconsisting of 90% ACN and 10% water, 95% ACN and 5% water, 98% ACN and2% water, respectively, and a flow rate of 1.0 mL/minute. Drug contentwas determined relative to peak areas from drug standards (0.05-50μg/mL) in methanol.

Measures of Drug Formulation Aqueous Solubility

The aqueous solubility of CAB, MCAB, M2CAB and M3CAB in optima water wasdetermined by adding the excess drug or prodrug powder in 1 mL water tomake the saturated aqueous solution. The mixture was stirred for 24hours at room temperature. Further, the solution was centrifuged at14000 rpm for 10 minutes to pellet the undissolved powder. Thesupernatant was collected, lyophilized and dissolved in methanol for thedrug content analysis by UPLC UV/Vis.

Chemical Stability

The stability of MCAB, M2CAB and M3CAB in acidic (pH 1), basic (pH 11)and neutral (pH 7) conditions at room temperature and elevatedtemperature (37° C.) was determined. The stock solution of each prodrugwas prepared in DMSO at a concentration of 1 mg/mL. For acidic, basicand neutral assays, 100 μL of stock solution of each prodrug was addedto 1900 μL of 0.1 M HCl, 0.1 M NaOH or optima-grade water (pH adjustedto 7), respectively. Samples were then incubated at room temperature and40° C. under shaking conditions (Innova® 42 shaker incubator, 150 rpm).Samples were withdrawn at 0, 2, 4, 8 and 24 hours and stored at −80° C.Later, samples were analyzed for drug content by UPLC-UV/Vis.

Plasma Cleavage Drug Hydrolysis Kinetics

The hydrolysis kinetics of MCAB, M2CAB and M3CAB and relative release ofactive drug were determined in plasma of different species (mouse, rat,rabbit, monkey, dog, and human). MCAB, M2CAB or M3CAB (1 μM) wereincubated in 100 μL plasma at 37° C. At different time points, 1 mLacidified methanol (0.1% formic acid and 25 mM ammonium formate to avoidfurther prodrug hydrolysis) was added to each sample and vortexed for 3minutes to stop the reaction. For 0-minute time-point, a 100 μL ice coldplasma was spiked with prodrug stock solution, and 1 ml of ice coldacidic methanol was added immediately. Following the addition ofmethanol, samples were centrifuged at 15,000 g for 10 minutes, andcollected supernatant was analyzed for drug content by UPLC-MS/MS(Waters Xevo TQ-XS).

Nanoparticle Synthesis and Characterization

Nanoformulations of the parent CAB (NCAB) and of its prodrugs (NMCAB,NM2CAB and NM3CAB) were manufactured by high-pressure homogenizationusing the poloxamer surfactant, 407 (P407). The drug/prodrug and P407were premixed (10:1 w/w) in endotoxin free water for 24 hours in theconcentration range of 2%-20% w/v drug/prodrug and 0.2-2% w/v P407. Thepremix was further homogenized using an Avestin EmulsiFlex-C3high-pressure homogenizer (Ottawa, ON, Canada) at 18,000 psi to generatehomogenous nanocrystals of desired particle size. Nanoparticles werecharacterized for particle size (D_(eff)), polydispersity index (PDI),and zeta potential by dynamic light scattering (DLS) using a MalvernNano-ZS (Worcestershire, UK). The stability of the nanoformulations wasmonitored at 4, 25 and 37° C. over 3 months. Drug/prodrug content innanoformulation was measured by dissolving the nanoformulation inmethanol (Dilution factor range: 1000-10000), and analyzed by UPLCUV/Vis. Following equation was used to calculate encapsulationefficiency. Encapsulation efficiency (%)=(weight of drug informulation/initial weight of drug added)×100. Morphology ofnanoparticles was assessed by scanning electron microscopy (SEM).Nanoparticles were fixed in a solution of 2% glutaraldehyde, 2%paraformaldehyde in a 0.1 M sorenson's phosphate buffer (pH 7.2) at 4°C. for 24 hours and processed for imaging. Briefly, nanosuspensions wereair dried onto a glass coverslip mounted on an SEM sample stub andsputter coated with approximately 50 nm of gold/palladium alloy. Sampleswere assayed using a FEI Quanta 200 scanning electron microscope(Hillsboro, Oreg.) operated at 5.0 kV (Sillman, et al., Nat. Commun.(2018) 9:443).

Human Monocytes Derived Macrophages (MDM)

Human monocytes were obtained by leukapheresis from HIV-1/2 andhepatitis B seronegative donors and later purified by counter-currentcentrifugal elutriation (Gendelman, et al., J. Exper. Med. (1988)167:1428-1441). Monocytes were cultured in DMEM media containing 4.5 g/Lglucose, L-glutamine, and sodium pyruvate supplemented with 10%heat-inactivated human serum, 50 μg/mL gentamicin, and 10 μg/mLciprofloxacin. Cells were maintained at 37° C. in a 5% CO₂ incubator.Recombinant human macrophage colony stimulating factor (MCSF, 1000 U/mL)was added to culture media for first 7 days to facilitatemonocyte-derived macrophages (MDMs) differentiation. Half-culture mediawas replaced with fresh media every other day. After differentiation,MDM were utilized for following in vitro assays.

Cytotoxicity Assay

Cellular viability following treatment with nanoparticles was evaluatedby performing MTT assay. Human MDM plated in 96-well plates at a densityof 0.08×10⁶ cells per well were treated with 10, 25, 50, 100, 200, or400 μM NCAB, NMCAB, NM2CAB or NM3CAB for 24 hours. Untreated cells wereused as controls. For each group samples were in quadruplets. Cells werewashed with PBS and incubated with 100 μL/well of MTT solution (5 mg/mL)for 45 minutes at 37° C. After incubation, MTT solution was removed, andcells were washed with PBS. Then, 200 μL of DMSO was added to each well,and absorbance was measured at 490 nm on a Molecular Devices SpectraMax®M3 plate reader with SoftMax Pro 6.2 software (Sunnyvale, Calif.).

Studies of Drug Particle MDM Uptake, Retention and Release

MDM uptake and retention studies were performed in clear flat-bottom12-well plates at a density of 1.0×10⁶ cells per well, with eachtreatment group completed in triplicate. For cellular uptake studies,MDM were treated with 100 μM NCAB, NMCAB, NM2CAB or NM3CAB. MDM werecollected at 2, 4, 8 and 24 hours following treatment to measureintracellular drug and prodrug levels. For retention studies, MDM weretreated with 100 μM of NCAB, NMCAB, NM2CAB, or NM3CAB for 8 hours, andthen washed twice with phosphate buffered saline (PBS). Fresh culturemedium was added and half-media was replaced every other day. MDM werecollected at days 1, 5, 10, 15, 20, 25 and 30 to assay intracellulardrug and prodrug concentrations. For both studies, at stated timepoints, adherent MDM were washed twice with PBS. Then cells were scrapedinto PBS, and counted using an Invitrogen Countess™ Automated CellCounter (Carlsbad, Calif.). Cells suspension in PBS was centrifuged at3,000 rpm for 8 minutes at 4° C. Obtained cell pellets were sonicated in200 μL methanol using a probe sonicator to extract drug to extractintracellular drug. The resultants were centrifuged at 20,000 g for 10minutes at 4° C. to separate cell debris from drug containingsupernatant. Samples were further analyzed for drug and prodrug contentsby UPLC-UV/Vis. For release studies, culture medium at the time pointssimilar to those in the retention study was collected for quantitatingthe drug and prodrug released by MDM. The culture medium was mixed withmethanol to precipitate the non-soluble components in the culture mediumand to extract drug and prodrug. The mixture was centrifuged at 17,000 gfor 10 minutes at 4° C. to separate the non-soluble precipitate. Thesupernatant was transferred to new tubes to be dried in speed vacuum.The dried contents were suspended in methanol for further analyzed byUPLC-UV/Vis.

Particle Characterization by Transmission Electron Microscopy (TEM)

MDM were treated with NCAB, NMCAB, NM2CAB or NM3CAB at concentration of100 μM for 8 hours, and then washed twice with PBS. Fresh culture mediawere added and half-media was replaced every other day. MDM culturesupernatant fluids were collected at days 0, 15, and 30 afterdrug-particle treatment, and analyzed by TEM to image intracellularnanoparticles. For day 0, cells were collected right after 8 hourstreatment duration. At stated time points, cells were washed, scrapedinto PBS, pelleted at 3000 rpm for 8 minutes at room temperature, andfixed in a solution of 2% glutaraldehyde, 2% paraformaldehyde in 0.1 MSorenson's phosphate buffer (pH 6.2). A drop of the fixed cellsuspension was placed on a formvar/silicon monoxide 200 mesh copper gridand allowed to settle for 2 minutes. The excess solution wicked off andallowed to dry. A drop of NanoVan vanadium negative stain was placed onthe grid for 1 minute, then wicked away and allowed to dry. Grids wereexamined on a FEI Tecnai G2 Spirit TWIN transmission electron microscope(Hillsboro, Oreg.) operated at 80 kV. Images were acquired digitallywith an AMT digital imaging system (Woburn, Mass.) (Sillman, et al.,Nat. Commun. (2018) 9:443).

HIV-1 Infection and Measurements of Reverse Transcriptase (RT) Activityin Infected Cell Fluids

MDMs were plated in clear flat-bottom 24-well plates at a density of0.8×10⁶ cells/well. MDM were treated with 100 μM of NCAB, NMCAB, NM2CABor NM3CAB for 8 hours. Following treatment, cells were washed with PBSand cultured in fresh culture medium with half-media replacement everyother day. At 1, 5, 10, 15, 20, 25 and 30 days after the treatment, thecells were infected with HIV-1ADA (a macrophage tropic viral strain) ata multiplicity of infection (MOI) of 0.1 infectious particles per cellfor 16 hours. Following infection period, MDM were washed with PBS andreplenished with fresh media. Cells were cultured for next ten days withhalf-media replacement every other day, and the full media replacementon the 8th day. The culture media was collected on the 10th day afterinfection for the measurement of HIV-1 RT activity (Kalter, et al., J.Immunol. (1991) 146:3396-3404; Nowacek, et al., J. Neuroimmune Pharm.(2010) 5:592-601). The extent of infection was expressed as a percent ofRT activity by infected MDM that were not treated. Cells were fixed in2% PFA, and expression of HIV-1 p24 antigen was determined byimmunocytochemistry (Nowacek, et al., J. Neuroimmune Pharm. (2010)5:592-601).

Half-Maximum Effective Concentration (EC₅₀) Assay

MDM were plated in clear flat-bottom 96-well plates (0.08×10⁶cells/well). Cells were treated with a range of drug concentrations,0.01-1000 nM of CAB, MCAB, M2CAB, M3CAB, NCAB, NMCAB, NM2CAB, or NM3CABfor 1 hour prior to infection with HIV-1ADA (MOI of 0.1 infectiousparticles per cell) for 4 hours. After 4 hours of viral challenge, cellswere washed and given fresh media containing drug (0.1-1000 nM).Subsequently, cell supernatants were collected 10 days later and assayedfor HIV-1 RT activity as described above.

Nanoparticle Uptake in CD4+ T-Cells Using CEM-ss Cells as Standards

CEM-ss CD4+ T-cells were suspended in RPMI supplemented with 10% fetalbovine serum, 100 U/mL penicillin and 100 μg/mL streptomycin. Cellsuspension (1 mL/well) was added in clear flat-bottom 12-well platespre-coated with poly-L-lysine solution (500 μg/mL in distilled water)for 1 hour. After attachment to wells surface, cells were treated with25 μM NCAB, NMCAB, NM2CAB or NM3CAB. At 2, 4 and 8 hours, cells werewashed and scrapped into PBS. Cell suspension was centrifuged at 200 gfor 5 minutes and intracellular drug and prodrug concentrations werequantified by Waters TQD mass spectrometer. Uptake of nanoparticles inCEM-ss CD4+ T-cell lines was confirmed by TEM imaging after 8 hours oftreatment at 25 μM concentration. Sample processing for TEM imaging isdescribed above.

Pharmacokinetics (PK) and Biodistribution (BD) Mouse Studies

NSG mice (Female, 6-8 weeks, Jackson Labs, Bar Harbor, Me.) wereadministered 45 mg/kg CAB-equivalents of NCAB, NMCAB, NM2CAB or NM3CABby a single intramuscular (IM, caudal thigh muscle) injection at 40μL/25 g mouse. Following injection, blood samples were collected intoheparinized tubes at day 1 post-administration and then weekly up to day364 by cheek puncture (submandibular vein, MEDIpoint, Inc., Mineola,N.Y.). Collected blood (25 μL) was immediately diluted into 1 mL ACN andstored at −80° C. until drug measurements. Remaining blood samples werecentrifuged at 2,000 g for 8 minutes for plasma collection. Plasma wascollected and stored at −80° C. for analysis of drug contents. At day14, 28, 42 and 364 following administration, animals were humanlyeuthanized by isoflourane inhalation, followed by cervical dislocation.Spleen, lymph nodes, liver, lung, gut, kidney, brain, vaginal tissue,and rectal tissue were collected for quantitation of CAB and prodrugconcentrations. CAB, MCAB, M2CAB and M3CAB were quantitated in mouseplasma, blood and tissues by UPLC-MS/MS using a Waters ACQUITY H-classUPLC (Waters, Milford, Mass., USA) connected to a Xevo TQ-S micro massspectrometer. All solvents for sample processing and UPLC-MS/MS analysiswere LCMS-grade (Fisher). For plasma and blood samples, 25 μL of samplewas added into 1 mL acetonitrile (ACN) spiked with 10 μL internalstandard (IS). d3-Dolutegravir (d3-DTG), myristoylated dolutegravir(MDTG), and stearoylated darunavir (SDRV), at a final concentration of200, 20 and 20 ng/mL, respectively, were used as ISs for CAB, MCAB andM2CAB/M3CAB analyses, respectively. Samples were vortexed andcentrifuged at 17,000×g for 10 minutes at 4° C. The supernatants werecollected and dried using a SpeedVac® and reconstituted in 100 μL 80%methanol; 10 μL was injected for MCAB, M2CAB, and M3CAB UPLC-MS/MSanalysis. Standard curves were prepared in blank mouse plasma/blood inthe range of 0.2-2000 ng/mL for CAB, MCAB, M2CAB, and M3CAB. For tissuedrug quantitation, 3-200 mg of sample was homogenized in 4-29 volumes of0.1% v/v formic acid and 2.5 mM ammonium formate containing 90%methanol. To 100 μL of tissue homogenate was added 280 μl methanolcontaining 0.1% formic acid and 2.5 mM ammonium formate, 80% methanol(10 μL), and IS (10 μL), followed by vortexing for 3 min andcentrifugation at 17,000×g for 15 minutes. For MCAB, M2CAB and M3CABanalyses, 85 μl of supernatant was mixed with 15 μl water. For CABanalysis, 20 μl of supernatant was mixed with 80 μl of 50% ACN. Thesealiquots were vortexed, centrifuged at 17,000×g for 10 minutes and 10 μlof supernatant was used for LC-MS/MS analysis. Standards were preparedsimilarly using blank tissue homogenates with 10 μL of spiking solution(CAB/MCAB/M2CAB/M3CAB, 5-20,000 ng/mL in 50% ACN). For CAB quantitation,chromatographic separation of 10 μL CAB sample was performed on a WatersACQUITY UPLC BEH Shield RP18 column (1.7 μm, 2.1 mm×100 mm) using a10-minute gradient of mobile phase A (7.5 mM ammonium formate in water,adjusted to pH 3 using formic acid) and mobile phase B (100% ACN) at aflow rate of 0.25 mL/minute. For the first 3.5 min, the mobile phasecomposition was 35% B and was increased to 95% B in 0.5 min and heldconstant for 1.5 minute. Mobile phase B was then reset to 35% in 0.5 minand the column was equilibrated for 1 minute before the next injection.For MCAB and M2CAB quantitation chromatographic separation was achievedon a shorter 30 mm column (PN with an 8-min gradient method at a flowrate of 0.28 mL/minute. The initial mobile phase composition was 80% Bfor the first 2 min, and increased to 95% B in 4 minutes, held constantfor 0.75 minute, reset to 80% in 0.25 minute and the column wasequilibrated for 1 minute before the next injection. For M3CABquantitation chromatographic separation was achieved also on a shorter30 mm column with an 8-minute gradient method at a flow rate of 0.35mL/min. The initial mobile phase composition was 88% B for the first 5min, and increased to 95% B in 0.25 min, held constant for 1.5 minutes,reset to 88% in 0.25 minute and the column was equilibrated for 1 minutebefore the next injection. CAB, MCAB, M2CAB, and M3CAB were detected ata cone voltage of 10 V, 24 V, 2 V and 20 V respectively, and a collisionenergy of 24 V, 18 V, 24 V and 26 V respectively, in the positiveionization mode. Multiple reaction monitoring (MRM) transitions used forCAB, MCAB, M2CAB, M3CAB, d3-DTG, MDTG and SDRV were 406.04>126.93,616.28>406.09, 672.34>406.07, 728.47>406.09, 422.84>129.99,630.20>420.07 and 814.52>658.44, respectively. Spectra were analyzed andquantified by MassLynx software version 4.1. All quantitations weredetermined using analyte peak area to internal standard peak arearatios. Following PK and BD analysis in NSG mice, superior NM2CAB, amongall formulations, was again evaluated in another strain of mice,BALB/cJ. (Male, 6-8 weeks, Jackson Labs). NCAB was used as a control.Mice injected with NM2CAB were humanly euthanized at day 280 followingadministration. Drug and prodrug quantitation in collected plasma andtissue was similar to above. Non-compartmental PK analysis for plasmaCAB was performed using Phoenix WinNonlin-8.0 (Certara, Princeton, N.J.)for studies in NSG mice.

PK and BD in rhesus monkeys (RM)

Four male RMs (4.4-6.7 kg; PrimeGen) were anesthetized with ketamine (10mg/kg) and subsequently administered 45 mg/kg CAB-eq. of NM2CAB and alab-generated RPV prodrug by IM injection (quadriceps femoris muscle,0.5 mL/kg). Blood samples were collected in potassium-EDTA coated tubesfor complete blood counts (CBC), metabolic profiles. Plasma wasseparated for drug measurements. Tissue biopsies of lymph node, adiposeand rectal tissues were collected at day 204 after injection for drugquantitation. Quantitation methods for drug and prodrug in plasma andtissues were similar to described above in mice studies.

Statistical Analyses

Statistical analysis were made using GraphPad Prism 7.0 software (LaJolla, Calif.). Data of in vitro studies were expressed as mean±SEM witha minimum of 3 biological replicates, while in vivo study results wereexpressed as mean±SEM with a minimum of 3 biological replicates. Forcomparisons between two groups, Student's t test (two-tailed) was used.A one-way ANOVA followed by a Tukey's test was used to compare three ormore groups. Statistical significances were denoted as follows: *P<0.05,**P<0.01, ***P<0.001, ****P<0.0001.

Study Approvals

All animal studies were approved by the University of Nebraska MedicalCenter Institutional Animal Care and Use Committee (IACUC) in accordancewith the standards incorporated in the Guide of the Care and Use ofLaboratory Animals (National Research Council of the National Academies,2011). Human monocytes were isolated by leukopheresis from HIV-1/2 andhepatitis B seronegative donors according to an approved UNMCInstitutional Review Board exempt protocol.

Results

Prodrug Synthesis and Characterization

CAB was chemically modified by attaching fatty acid chains of variablecarbon lengths-14, 18, and 22, to produce esters MCAB, M2CAB, and M3CABrespectively. The prodrugs were further characterized by nuclearmagnetic resonance (NMR) to confirm the synthesis. ¹H NMR spectra of allthree prodrugs showed triplets in the range of 0.86-0.89, 1.77-1.78 and2.66-2.70 ppm and a broad singlet in the range of 1.24-1.25 ppmcorresponding to the terminal methyl and repeating methylene protons ofthe fatty acid alkyl chain. Disappearance of the phenol proton peak at11.5 ppm confirmed the substitution of CAB's hydroxyl proton with fattyacyl moieties. ¹³C NMR spectrum of each prodrug confirmed the carbonatoms of the conjugated aliphatic chain. Electrospray ionisation massspectrometry (ESI-MS) affirmed the molecular weights of all prodrugs.ESI-MS infusion for MCAB generated a strong signal at 616.28 m/z, ESI-MSinfusion for M2CAB generated a strong signal at 672.34 m/z, and ESI-MSinfusion for M3CAB generated a strong signal at 728.47 m/z. Fouriertransform infrared spectroscopy (FTIR) prodrug characterization producedbands not observed for CAB in the ranges of 2908-2935 and 1748-1775cm⁻¹. These correspond to C—H stretches in aliphatic methylene groupsand C═O stretch corresponding to carboxyl group in ester bond,respectively. XRD spectra over 20=2-70° showed the crystalline characterof prodrugs MCAB, M2CAB and M3CAB. Nanoparticles of M2CAB maintained thecrystalline property of prodrug. A significant serial reduction in theaqueous solubility of MCAB (2.83±1.37 μg/mL), M2CAB (1.91±0.23 μg/mL)and M3CAB (1.51±0.7 μg/mL) was observed compared to CAB (12.12±0.41μg/mL). Moreover, compared to CAB (0.09±0.002 mg/mL), a parallelsignificant increase in 1-octanol solubility of MCAB (2.31±28.15 mg/mL),M2CAB (2.64±0.02 mg/mL) and M3CAB was observed. These results confirmedthe increase in the hydrophobicity and lipophilicity of the prodrugscompared to CAB as a result of chemical modifications with the fattyacid conjugates.

Prodrugs are pharmacologically inactive compounds that require enzymaticor hydrolytic activation for bioconversion into active drugs inphysiological conditions. Therefore, hydrolysis kinetics of MCAB, M2CABand M3CAB and subsequent CAB formation were evaluated in plasma ofdifferent species (mouse, rat, rabbit, monkey, dog, and human). Uponincubation in plasma of all the species tested, MCAB showed more than85% cleavage within 30 minutes, M2CAB showed an average of 75% cleavageby 2 hours and 80% cleavage by 6 hours. M3CAB showed the slowest rate ofcleavage with around 50% prodrug remaining after 24 hours of incubationin plasma. These studies showed that MCAB rapidly converted into CABwhile M3CAB showed the least conversion into CAB. CAB formation fromM2CAB hydrolysis was an average 71.1% by 6 hours, signifying that M2CABis the optimal prodrug in terms of stability and active drug formationin physiological conditions. There were some differences in rates ofprodrug hydrolysis among plasma of various animal species. Thevariability in the plasma CAB concentrations amongst the tested speciescan be the result of differences in body fat distribution, muscles mass,physical activity and in plasma carboxylesterases enzymes operative forprodrug hydrolysis (Trezza, et al., Curr. Opin. HIV AIDS (2015)10:239-245; Bahar, et al. (2012) J. Pharm. Sci. (2012) 101:3979-3988;Wang, et al., Acta Pharmaceutica Sinica. B (2018) 8:699-712). Moreover,chemical stability of M2CAB was determined at room temperature andelevated temperature (37° C.) in acidic (pH 1), neutral (pH 7), andbasic conditions (pH 11) over 24 hours to determine the long-termstability of prodrug nanoformulations at various storage conditions. Atroom temperature, at 24 hours, 19% hydrolysis in acidic conditions, and24% hydrolysis in neutral conditions were determined. However, at basicconditions M2CAB was completely hydrolyzed and was undetected. At 37°C., at 24 hours, around >95% prodrug was hydrolyzed in acidic conditionsand 28% prodrug was hydrolyzed in neutral conditions. Whereas, at basicconditions M2CAB was completely hydrolyzed within 2 hours. Thehalf-maximum effective concentration (EC₅₀) values of the prodrugs,MCAB, M2CAB and M3CAB, were compared with CAB to determine the changes,if any, in antiretroviral activity due to chemical modification. HIV-1RT activity measurements of the culture medium of MDM infected withHIV-1_(ADA) demonstrated that the EC₅₀ values of CAB (28.39 nM), MCAB(34.19 nM), M2CAB (44.71 nM), and M3CAB (51.15 nM) were comparative,indicating no significant effect on drug activity.

Nanoformulation Manufacture and Characterization

Nanoformulations of CAB (NCAB), MCAB (NMCAB), M2CAB (NM2CAB) and M3CAB(NM3CAB) were manufactured by top down synthesis utilizing high-pressurehomogenization. The encapsulation efficiency of drug or any of prodrugwas >85%. Poloxamer 407 (P407) surfactant provided particle surfacestabilization. Nanoparticle size, polydispersity index (PDI), and zetapotential were determined by DLS at room temperature (RT), 4° C., and37° C. All the formulations remained stable up to 98 days, signifyingthat these formulations can maintain their physical integrity at a rangeof storage conditions. Moreover, temperature variation did not affectthe physicochemical properties of any formulation over the period ofstudy. At the day of manufacture, average particle size, PDI and zetapotential for NCAB were 294.5±1.8 nm, 0.23±0.01, −28.3±0.21 mV; forNMCAB were 302.1±10.8 nm, 0.23±0.01, −31.1±1.1 mV; for NM2CAB were359.7±3.63 nm, 0.23±0.02, −31.3±0.1 mV; and for NM3CAB were 268.47±6.9nm, 0.23±0.03, −31.1±0.06 mV. At day 98 post-manufacture,physicochemical parameters for NCAB were 327.5±4.9 nm, 0.21±0.04,−18.2±0.25 mV; for NMCAB were 388.7±6.4 nm, 0.22±0.03, −24.8±2.87 mV;for NM2CAB were 369.5±2.5 nm, 0.19±0.03, −19.6±0.53 mV; and for NM3CABwere 245.6±2.6 nm, 0.27±0.06, −25.7±0.56 mV. The SEM images showed thatNCAB, NMCAB, NM2CAB and NM3CAB nanoparticles had uniform rod-shapedmorphology. For conformation of reproducibility, the NM2CAB formulationwas manufactured in 11 separate batches (Table 1). Nanoparticle sizesvaried from 243.00±2.48 to 378.00±1.90 nm with a narrow PDI (0.18±0.03to 0.33±0.03).

TABLE 1 Reproducibility of NM2CAB synthesis Manufacture Batch no. Size ±SEM (nm) Pdl ± SEM 1 320.13 ± 6.81 0.23 ± 0.02 2 322.10 ± 2.99 0.21 ±0.01 3 328.67 ± 1.60 0.18 ± 0.03 4 311.43 ± 4.53 0.33 ± 0.03 5 287.17 ±0.94 0.22 ± 0.01 6 317.57 ± 2.83 0.27 ± 0.01 7 378.00 ± 1.90 0.26 ± 0.018 244.63 ± 5.85 0.19 ± 0.01 9 268.97 ± 7.66 0.19 ± 0.02 10 259.77 ± 2.090.23 ± 0.01 11 243.00 ± 2.48 0.22 ± 0.01

The effect of nanoformulations on antiretroviral activity of prodrugs(EC₅₀) was determined. Antiviral activity of NCAB, NMCAB, NM2CAB, orNM3CAB was determined in MDM at a range of concentrations (0.01-1000 nM)and measured by HIV-1 reverse transcriptase activity after viralchallenge with HIV-1ADA at an MOI of 0.1. EC₅₀ values were increasedcompared to non-nanoformulated drug or prodrugs, likely due to therequired dissolution of nanoparticles prior to cleavage of prodrugs.EC₅₀ values were comparable among NCAB (39.83 nM), NMCAB (89.67 nM),NM2CAB (37.02 nM). However, the EC₅₀ value for NM3CAB was increasedsignificantly (˜1.78E+06 nM). This could be an effect of slower cleavageof prodrug and subsequent generation of active CAB as well as ofintracellular stability of nanoformulations. For the furthercharacterization of prodrug nanoformulations and determination of thetreatment concentrations for in vitro studies, the cellular vitality wasassessed in MDM and CEM-ss CD4+ T-cells by MTT assay. In MDM, nocytotoxicity was seen at the tested range of concentrations (10-400 μM)for all nanoformulations. In CEM-ss CD4+ T-cells, cytotoxicity wasdetermined at 50 μM and above concentrations. Therefore, treatmentconcentrations for all nanoformulations were 100 μM for assays in MDMand 25 μM for studies in CEM-ss CD4+ T-cells.

In Vitro Screening in MDM and CEM-Ss CD4+ T Cells

Macrophages can be successfully utilized as cellular drug depots andcarriers. Because of their phagocytic nature and the ability to migratethroughout the body (Zhou, et al., Biomaterials (2018) 151:53-65;Darville, et al., J. Pharm. Sci. (2014) 103:2072-2087; Aderem, et al.,Ann. Rev. Immunol. (1999) 17:593-623; Dou, et al., Blood (2006)108:2827-2835), MDM can serve as drug delivery systems to viralreservoirs. Therefore, MDM were used as in vitro system to evaluate thenanoformulations (Zhou, et al., Biomaterials (2018) 151:53-65; Hilaire,et al., J. Control Release (2019) 311-312:201-211; Ibrahim, et al., Int.J. Nanomedicine (2019) 14:6231-6247; Lin, et al., Chem. Commun. (2018)54:8371-8374; Sillman, et al., Nat. Commun. (2018) 9:443; Smith, et al.,Biomaterials (2019) 223:119476; Soni, et al., Biomaterials (2019)222:119441).

Uptake assay was performed in MDM by measuring drug and prodrug levelsfollowing treatment with 100 μM NCAB, NMCAB, NM2CAB or NM3CAB up to 24hours. Intracellular prodrug levels measured for NMCAB, NM2CAB andNM3CAB were 61.69±0.78, 84.07±5.82, and 73.34±13.59 nmoles/10⁶ cells,respectively by 24 hours; and intracellular CAB levels were 0.58±0.11,12.31±0.46, 17.79±2.92, and 7.97±1.76 nmoles/10⁶ cells for NCAB, NMCAB,NM2CAB or NM3CAB, respectively. Afterwards, the capacity of macrophagesto retain intracellular drug and prodrug was evaluated over a period of30 days following single treatment. MDM were treated with 100 μM NCAB,NMCAB, NM2CAB or NM3CAB for 8 hours. Intracellular prodrug levels weresustained up to 30 days following single treatment. Specifically, theamount of intracellular prodrug measured at day 30 for NMCAB, NM2CAB andNM3CAB were 0.41±0.09, 14.21±2.28, and 26.70±3.29 nmoles/10⁶ cells.Similarly, intracellular CAB levels formed from prodrug cleavage weremeasured up to 30 days. The amount of intracellular CAB levels measuredat day 30 for NM2CAB and NM3CAB were 1.71±0.35 and 2.05±0.10 nmoles/10⁶cells. Whereas, intracellular CAB levels fell below the limit ofquantitation within 24 hours after NCAB treatment; and intracellular CABconcentration following NMCAB treatment was measured up to day 25(0.09±0.04 nmoles/10⁶ cells), and was undetectable at day 30. Inparallel to retention assay, CAB released into culture fluids weremeasured over 30 days. NM2CAB showed the most sustained CAB release withdrug levels of 1.0±0.10 nmoles/10⁶ cells at day 30. No CAB was detectedwith NCAB and NM3CAB treatment. Prodrugs were not detected in culturemedium for any of the treatment, indicating prodrug bioconversion.

Next, TEM images of MDM were taken at day 0, 15, 30 after treatment withnanoformulations for 8 hours to assess the presence of nanoparticles inthe cytoplasmic vesicles. TEM images confirmed the presence of prodrugnanoformulations (NM2CAB and NM3CAB) up to day 30 in MDM, signifying thedrug-depot property of MDM; and validated the uptake, retention andrelease results. Uptake of NCAB, NMCAB, NM2CAB or NM3CAB was determinedin CD4+ T-cells following treatment at 25 μM concentration reflectedwhat was observed in MDM. TEM imaging performed on CEM-ss CD4+ T-cellsfollowing single treatment of NCAB, NMCAB, NM2CAB or NM3CAB for 8 hoursconfirmed the presence of nanoparticles in cytoplasmic compartments.

To examine whether sustained drug retention in MDM would protect againstHIV-1 infection, cells were challenged with HIV-1ADA up to 30 daysfollowing an 8-hour treatment with 100 μM NCAB, NMCAB or NM2CAB andassayed quantitatively for HIV-1 RT activity, as well as qualitativelyfor HIV-1 p24 antigen expression. NM3CAB was not selected for this studyas it did not show significant protection at the desired EC₅₀ value.NM2CAB treatment suppressed HIV-1 RT activity up to day 30 and wasconfirmed by absence of HIV-1 p24 expression. In contrast, completeviral breakthrough occurred at day 1 post-NCAB treatment and at day 20post-NMCAB treatment. Therefore, enhanced MDM drug retention exhibitedby NM2CAB provided superior protection against HIV-1 challenge comparedto NACB and NMCAB. In addition, dose response antiretroviral activity ofNM2CAB was determined (FIG. 4). MDM were treated with 10, 50 or 100 μMNM2CAB for 8 hours and challenged with HIV-1ADA. Similar to above,antiretroviral activity was determined up to 30 days. A complete viralsuppression was observed in 50 and 100 μM NM2CAB treatments while 57%protection was seen in 10 μM treatment after 30 days and validated byHIV-1 p24 expression (FIG. 4).

Pharmacokinetics (PK) and Biodistribution

To assess PK and biodistribution profiles, female NSG mice were injectedIM with single dose of 45 mg/kg CAB-equivalents of NCAB or NMCAB orNM2CAB. Immunodeficient female NSG mice were used for evaluation tomimic disease pathology of immune compensation and to assessbiodistribution in genitourinary tracks. NCAB is an effective equivalentformulation to CAB-LA as both formulations yielded similar plasma CABlevels up to day 49 after administration to BALB/cJ mice.

At day 1 post-injection, NCAB treatment generated higher plasma CABconcentrations compared to both NMCAB and NM2CAB and showed faster decaykinetics over the study period in comparison to NM2CAB. With NCABtreatment, plasma CAB concentrations were maintained above the fourtimes protein-adjusted 90% inhibitory concentration (4×PA-IC₉₀; 664ng/mL) up to day 35 (792.7 ng/mL), then rapidly declined to below thePA-IC₉₀ (166 ng/mL) by day 49 (75 ng/mL) before falling below the limitof quantitation (0.5 ng/mL) by day 126. NMCAB treatment showed slowerdecay, and maintained plasma CAB levels above the 4×PA-IC₉₀ up to day 91(673.8 ng/mL) and above PA-IC₉₀ up to day 168 (186.7 ng/mL). At day 364post-NMCAB treatment, CAB levels were quantified at 8.5 ng/mL. In starkcontrast, NM2CAB treatment provided slower plasma decay kineticscompared to both NCAB and NMCAB up to day 364, maintaining sustainedplasma CAB concentration above the 4×PA-IC₉₀ up to day 231 (702 ng/mL)and above the PA-IC₉₀ up to day 364 (354.2 ng/mL). Plasmapharmacokinetic parameters for CAB were determined usingnoncompartmental analysis for all treatment groups. Quantitation of PKparameters demonstrated that apparent CAB half-life following NM2CAB(131.56 days) treatment was 17- and 3-fold greater than those of NCAB(7.80 days) and NMCAB (44.40 days), respectively. Similarly, CAB meanresidence time (MRT) of NM2CAB was 21-fold longer than NCAB (201.94 vs.9.79 days, respectively) and 7-fold longer than NMCAB (201.94 day vs.30.76 day, respectively).

NM2CAB also elicited significantly higher CAB tissue levels for up to ayear. Tissue biodistribution of CAB was assessed at day 14, 28, 42 and364 after single IM injection in vaginal tissue, rectal tissue, spleen,liver, gut, brain, kidney, lung, and lymph nodes-anatomical associatedtissues. Drug levels in lymph nodes were determined in anatomicalregions associated with lymph nodes only at day 28 and 364, due theirimmature state in immunodeficient NSG mice. Notably, MCAB levels werelower than M2CAB at day 14, 28 and 42 and were undetectable at day 364.For NM2CAB, at day 364, prodrug levels were 3414.8 ng/g (spleen), 909.8ng/g (liver), 52.7 ng/g (lung), 50.3 ng/g (brain), 3.9 ng/g (kidney) and18710.1 ng/g (lymph nodes). At day 28, CAB levels in all tissues werecomparable between NCAB and NM2CAB. However, by day 42, CAB levels inall tissues tested after NCAB treatment were significantly lowercompared to those after NM2CAB treatment (vaginal tissue, spleen, gut,brain, kidneys and lungs rectal tissue and brain). Whereas, CAB levelsin tissues, up to day 42, following NMCAB treatment were significantlyhigher compared to NCAB and NM2CAB treatments. At day 364 followingtreatment with NM2CAB, CAB concentrations were significantly higher inall tested tissues compared to other formulations, and CAB levels weremeasured at 27 ng/g (vaginal tissue), 19.7 ng/g (rectum), 41.1 ng/g(spleen), 67.62 ng/g (lymph nodes-anatomical associated tissues), 123.9ng/g (liver), 10.3 ng/g (gut), 7.5 ng/g (brain), 33.2 ng/g (kidney) and35.5 ng/g (lung). In contrast, CAB levels were significantly low orbelow the limit of quantitation (0.5 ng/g) in all tissues at day 364after treatment with NCAB or NMCAB.

Prodrug (MCAB or M2CAB) concentrations in blood and all tissues was alsotested (FIG. 5). Concentrations of both prodrugs in blood were lower atday 1 post-injection, 22 ng/mL for MCAB and 31.3 ng/mL for M2CAB, andrapidly went blow the limit of quantitation, signifying bioconversion ofprodrugs to active parent drug. All the screened tissues weresubstantial depots for M2CAB and had sustained levels of M2CABthroughout the study period. Single IM injection of NM3CAB (45 mg/kgCAB-equivalents) in female NSG mice generated low levels of plasma CAB(2248 ng/mL) at day 1-post administration compared to NCAB (41237.6ng/mL), NMCAB (30148.9 ng/mL) and NM2CAB (7076.1 ng/mL). Plasma CABlevels reached around PA-IC₉₀ (233.2 ng/mL) within 28 days aftertreatment. Plasma and tissue levels of CAB and M3CAB were measured atday 28. For validation of slower hydrolysis of M3CAB, PK evaluation wasrepeated in BALB/cJ mice following single IM injection of NM3CAB at samedosage. Similar to PK results in NSG mice, NM3CAB generated low levelsof plasma CAB at day 1-post administration (777.8 ng/mL); and plasma CABlevels fell down below the one-time PA-IC₉₀ within 28 days (98.9 ng/mL).These data confirmed the slower prodrug hydrolysis of M3CAB prodrug andsubsequent bioconversion into the CAB.

Superior PK and BD profiles of NM2CAB among all formulations wereconfirmed in another strain of mice, BALB/cJ (male). Here, wild typemice were used to validate the results from immunodeficient NSG mice. PKand BD measurements in plasma and tissues for NM2CAB were parallel tothose in NSG mice. Particularly, plasma CAB concentrations were abovePA-IC₉₀ by day 231 (170.8 ng/mL) for NM2CAB, affirming the improvementin drug apparent half-life. NCAB was used as a control, in which CABlevels went below PA-IC₉₀ by day 28 (12.28 ng/mL).

To validate the results seen in rodents, rhesus macaques were injectedIM with single dose of 45 mg/kg CAB-equivalents of NM2CAB. Plasma CABand prodrug (M2CAB) levels were measured up to day 393. Similar toresults in mice, NM2CAB treatment provided slower plasma decay kinetics,maintaining sustained plasma CAB concentration up to day 393. At day393, CAB levels were measured at an average of 56.1 ng/mL. As expected,plasma M2CAB concentrations were lower throughout the study compared toCAB levels, signifying the bioconversion of prodrug to its active parentdrug (FIG. 6A). Rectal, lymph node, and adipose tissue biopsies werecollected at day 204 following NM2CAB administration and analyzed forCAB and M2CAB levels. CAB concentrations in the rectal, lymph node andadipose tissues were 10.12 ng/g, 22.7 ng/g and 29.5 ng/g, respectively(FIG. 6B). M2CAB was present at high levels in lymph node and adiposetissues (33.3 ng/g and 233.2 ng/g, respectively), with lower levels (1.7ng/g) in rectal tissue (FIG. 6C).

Toxicity Evaluation

Post-NM2CAB administration, toxicological assays were performed in bothmice and rhesus macaques. For toxicity assessment in NSG mice, animalweights were recorded weekly for a year; and at the study conclusion(day 364), plasma and tissues were collected for metabolic profiles andhistopathology, respectively. Age matched untreated mice were used ascontrols. No differences in weights were observed among animals from allgroups. Comprehensive serum chemistry parameters were quantified using aVetScan comprehensive diagnostic profile disc and a VetScan VS-2instrument. Examined parameters were (alanine aminotransferase (ALT),albumin (ALB), alkaline phosphatase (ALP), amylase (AMY) total calcium(CA++), creatinine (CRE), globulin (GLOB), glucose (GLU), phosphorus(PHOS), potassium (K+), sodium (NA+), total bilirubin (TBIL), totalprotein (TP), and urea nitrogen (BUN). No significant differences werenoted between controls and NM2CAB treated groups indicating that NM2CABdid not adversely affect functions of systemic organs. Histologicalexamination of tissue sections (liver, lung, gut, spleen, kidney andbrain) stained with H&E by a certified pathologist revealed no abnormalpathology in NM2CAB treated animals. Moreover, the formulation was welltolerated by both strains of mice (NSG and BALB/cJ), and no injectionsite reactions and changes in behavior or movement were observed.

For assessment in rhesus macaques, weights were recorded starting frompre-administration of formulation, and plasma and peripheral bloodmononuclear cells (PBMCs) were collected for complete blood counts andmetabolic profiles up to day 393 post-NM2CAB administration. Systemictoxicity was evaluated by measuring both hematologic (neutrophil,lymphocyte, monocyte) and metabolic (ALT, alkaline phosphatase,BUN/creatinine) profiles. No weight changes were recorded in any of theanimals following injection. An initial mild redness observed at thesite of injection was resolved by day 3 in all animals. No inflammationor bolus was noted 3 days after injection. Neutrophils, lymphocytes andmonocytes were counted prior to-and post-NM2CAB administration; and thecount of blood cells was consistent. At day 1 post-injection an increasein neutrophil count was recorded, and it became normal within 2 weeks inall animals. Such a change could be related to injection and may not bedrug associated. Liver and kidney metabolic profiles were unchanged inall animals following treatment. Overall, no adverse events wereobserved after NM2CAB administration.

Drug-Drug Interactions

Drug-drug interaction were evaluated between two prodrugnanoformulations (NM2CAB and NM3PRV) of drugs of different classes: CAB(INSTIs) and rilpivirine (RPV; NNRTI). RPV was a choice of drug alongwith CAB due to current clinical development of combination oflong-acting CAB and RPV nanoformulations. NM3RPV is a long-actingformulation of RPV (Hilaire, et al., J. Control Release (2019)311-312:201-211). BALB/cJ mice were treated IM with a single dose ofNM2CAB alone (45 mg/kg CAB-equivalents), NM3RPV alone (45 mg/kgRPV-equivalents) or co-administration of both prodrug nanoformulations(NM2CAB and NM3RPV, 45 mg/kg drug-equivalents). Plasma levels of CAB andRPV were measured, and no differences in active drug levels wereobserved between animals treated with formulations alone or incombination, signifying usage of combination of multiple prodrugnanoformulations for treatment or prevention.

With an inability to eradicate HIV infection protective vaccines andpre-exposure prophylaxis (PrEP) for persons at risk, long-term ART arethe sole approaches available to prevent disease and halt new infectionsand viral transmission. This is highlighted by ‘treatment as prevention’for those people at risk of infection. The focus in developing LA ARVinjectables center on creating drugs with long-half lives. For PrEP, LAARVs require “coverage” to preclude infections after HIV exposures.Viral infection need be halted during time periods where protective ARVlevels are detected in plasma. The drugs must also be given withoutuntoward side effects that include gastrointestinal toxicity and anynotable drug-drug interactions. Currently, LA agents have receivedenthusiasm amongst potential users with the potential advantages ofeliminating the stigma of viral infections and by requiringless-than-daily dosing intervals, some dosed as infrequently as every 2to 3 months. LA ARVs administered by subcutaneous or intramuscularroutes are highly effective and have already demonstrated enhanced lifequality and longevity (May, et al., AIDS (2014) 28:1193-1202;Antiretroviral Therapy Cohort, Lancet (2017) HIV 4:e349-e356). They helpcircumvent the lack of adherence to medication which remains as a keytreatment challenge (Shubber, et al., PLoS Med. (2016) 13:e1002183;Osterberg, et al., New Eng. J. Med. (2005) 353:487-497). Indeed, anypoor compliance to ARV regimens resulting in treatment failure,drug-resistant mutations, and new viral transmissions can be eliminatedwith improved adherence. In attempts to improve upon existing platformsfor LA ARVs, long acting slow effective release antiretroviral therapy(LASER ART) prodrugs have been developed. These new formulations serveto reduce frequency of injections while maintaining the sustainedtherapeutic levels of ARVs for longer duration (Edagwa, et al., Exp.Opinion Drug Del. (2017) 14:1281-1291). LASER ART comprises prodrugsynthesis through chemical modifications of existing ARV to provide slownative drug dissolution, poor water solubility, enhanced penetrancethough biological membranes of cell as well as tissue reservoirs, andlimited systemic off target toxicities. Prodrug synthesis allows theformation of ARV nanocrystals stabilized by polymer excipients. PK andefficacy (PD) of LASER ART formulations has been established for a rangeof ARVs including, but not limited, to dolutegravir (DTG), CAB, abacavir(ABC), lamivudine (3TC) and emtricitabine (FTC) (Zhou, et al.,Biomaterials (2018) 151:53-65; Hilaire, et al., J. Control Release(2019) 311-312:201-211; Ibrahim, et al., Int. J. Nanomed. (2019)14:6231-6247; Lin, et al., Chem. Commun. (2018) 54:8371-8374; McMillan,et al., Antimicrob. Agents Chemother. (2018) 62: e01316-17; McMillan, etal., AIDS (2019) 33:585-588; Sillman, et al., Nat. Commun. (2018) 9:443;Smith, et al., Biomaterials (2019) 223:119476; Soni, et al.,Biomaterials (2019) 222:119441).

CAB is an HIV-1 integrase strand transfer inhibitor (INSTI), and iscurrently is being developed as both oral and LA injectable (McPherson,et al., Expert Opin. Investig. Drugs (2018) 27:413-420). Its uniqueintrinsic properties, such as hydrophobic nature, long systemichalf-life (approximately 40 hours after oral administration), highpotency, resistant profile, low daily oral dosing requirement (<30mg/day), and limited drug-drug interactions, make it an attractivecandidate to develop into a LA injectable (Trezza, et al., Curr. Opin.HIV AIDS (2015) 10:239-245). Herein, it is demonstrated that a singleinjection of NM2CAB generated unexpectedly superior improvements in drugdurability reflective by sustained drug plasma concentrations and tissuebiodistribution compared against either NCAB, NMCAB, or NM3CAB. NM2CABprovided sustained plasma decay while maintaining drug levels above thePA-IC₉₀ of 166 ng/mL for 364 days following single injection.

CAB LA, currently nearing clinical approvals, was studied extensively inrhesus macaques affirming its abilities for use as an ARV injectable forpre-exposure prophylaxis (PrEP) studies (Edagwa, et al., Exp. Opin. DrugDel. (2017) 14:1281-1291; Stellbrink, et al., Curr. Opin. HIV AIDS(2018) 13:334-340). These studies demonstrated that CAB LA provided highextent of protection against vaginal, rectal, intravenous and penilechallenges with SHIV strains supporting its future use as for PrEP thosepeople at high-risk of HIV exposure and for intravenous drug users.Plasma levels above 3×PA-IC₉₀ provided 100% protection andconcentrations above PA-IC₉₀ provided 97% protection against viralchallenge (Edagwa, et al., Exp. Opin. Drug Del. (2017) 14:1281-1291;Stellbrink, et al., Curr. Opin. HIV AIDS (2018) 13:334-340). Herein,NM2CAB administration generated plasma CAB concentrations above PA-IC₉₀for more than six months signifying its superiority and its clinicallyefficacy as PrEP.

In all, the development of effective antiretroviral drug (ARV) treatmentand prevention measures for HIV-1-infected patients has changed fromwhat was certain death to a manageable life-long chronic ailment. Theneed for the control of the HIV-1 pandemic remains as an estimated 37.9people are infected worldwide. The advent of LA ARVs will certainlyexpand options for overcoming the challenge of suboptimal drug adherenceand reduce the burden of HIV infection. To date, chemoprophylaxis withHIV antiretroviral agents has been demonstrated in largest measure withTenofovir disoproxil fumarate (TDF)-containing compounds. However,required levels of adherence to daily or near-daily oral tablets hasproven challenging. LA preparations offer greater choice for achievingprevention with the understanding that safety, tolerability and efficacywill continue to be part of the therapeutic outcome assessments. LA ARVscapable of being administered on a monthly or less frequent basis willimprove therapeutic adherence and extend opportunities for PrEP.

A number of publications and patent documents are cited throughout theforegoing specification in order to describe the state of the art towhich this invention pertains. The entire disclosure of each of thesecitations is incorporated by reference herein.

While certain of the preferred embodiments of the present invention havebeen described and specifically exemplified above, it is not intendedthat the invention be limited to such embodiments. Various modificationsmay be made thereto without departing from the scope and spirit of thepresent invention, as set forth in the following claims.

What is claimed is:
 1. A compound of formula I:

or a pharmaceutically acceptable salt thereof, wherein R is a saturatedlinear aliphatic chain of a length of 17 carbons.
 2. A nanoparticle,comprising a compound of claim 1 and a polymer or surfactant.
 3. Thenanoparticle of claim 2, wherein said compound is

or a pharmaceutically acceptable salt thereof, and the polymer orsurfactant is P407.
 4. A pharmaceutical composition, comprising acompound of claim 1, and a pharmaceutically acceptable carrier.
 5. Amethod of treating an HIV infection in a subject in need thereof,wherein said method comprises administering to said subject atherapeutically effective amount a pharmaceutical composition of claim4.
 6. The method of claim 5, wherein the pharmaceutical composition isadministered via injection.
 7. The method of claim 5, wherein thepharmaceutical composition is administered one time in a 3, 6, 9, 12,18, or 24 month period.